TWI869334B - Lag-3 antibody pharmaceutical composition and use thereof - Google Patents

Abstract

The present invention relates to a LAG-3 antibody pharmaceutical composition and use thereof, wherein the LAG-3 antibody pharmaceutical composition comprises a LAG-3 antibody or antigen-binding fragment thereof in an acetate buffer or a histidine buffer. In addition, the pharmaceutical composition may also contain sugar, non-ionic surfactants and other excipients. The pharmaceutical composition of the present invention exhibits great antibody stability after storage for several months.

Description

A LAG-3 antibody pharmaceutical composition and its use

The present invention belongs to the field of pharmaceutical preparations. Specifically, the present invention relates to a pharmaceutical composition comprising a LAG-3 antibody and an antigen-binding fragment thereof, and its use as a drug.

Lymphocyte activation gene-3 (LAG-3, CD215) is a member of the immunoglobulin superfamily and can negatively regulate the functions and life cycle of immune cells. Studies have shown that LAG-3 plays an important role in immune system dysfunction caused by viral infection, autoimmune diseases and tumors. By affecting the function of LAG-3, the abnormal immune function in the evolution of these diseases can be improved, thereby improving the prognosis of the disease.

As a member of the immunoglobulin superfamily, LAG-3 consists of three parts: the extracellular region, the transmembrane region, and the cytoplasmic region. The mature LAG-3 molecule consists of 470 amino acids with a relative molecular weight of 70 kDa. It was first discovered by Triebel et al. in 1990 (J Exp Med, 1990, 171(5): 1393-405). Studies have found that LAG-3, like CTLA-4 and PD-1, is a negative co-stimulatory molecule, and its activation can negatively regulate lymphocyte function. LAG-3 is closely related to CD4 in structure, but has a completely opposite function to CD4, as shown by the high similarity between LAG-3 and CD4 molecules and the ability to bind to MHC-II (major histocompatibility complex) molecules. However, LAG-3 binds more tightly to MHC-II class molecules, thereby interfering with CD4 ⁺ T lymphocyte TCR activation and inhibiting T lymphocyte activation (Curr Opin Immunol, 2009, 21(2): 179-86; Eur J Immunol, 2003, 33(4): 970-9). In vitro studies have shown that LAG-3 can inhibit antigen-induced T lymphocyte proliferation responses. After blocking LAG-3, the activation and proliferation of T lymphocytes and the secretion of cytokines by type 1 T helper cells (Th1) were improved. Huang et al.'s study showed that the level of LAG-3 on the surface of activated CD4 ⁺ Treg cells was significantly increased, and LAG-3 was a necessary condition for CD4 ⁺ Tregs to exert the maximum immunosuppressive effect (Immunity, 2004, 21(4): 503-13). In addition, anti-LAG-3 antibodies can maintain the homeostasis of CD4 ⁺ and CD8 ⁺ T lymphocytes. After blocking LAG-3, the ability of CD8 ⁺ T lymphocytes to kill tumor cells is significantly enhanced (J Clin Invest, 2007, 117(11): 3383-92). Some disease-related studies have also found that LAG-3 plays an important role in regulating the occurrence and development of diseases. Gandhi et al. confirmed that the expression level of LAG-3 in T lymphocytes in human lymphoma tissues is related to T lymphocyte dysfunction, and the elimination of LAG-3 ⁺ T lymphocytes can significantly enhance the tumor clearance ability of lymphocytes (Blood, 2006, 108(7): 2280-9). The results of the study showed that LAG-3 is an important inhibitory molecule on the surface of immune cells and can have a significant negative regulatory effect on T lymphocytes.

LAG-3 is mainly expressed on T lymphocytes, B lymphocytes, NK cells, Treg cells and DCs (Proc Natl Acad Sci USA, 1997, 94(11): 5744-9. Eur J Immunol, 2005, 35(7): 2081-8; J Immunol, 2009, 182(4): 1885-91). LAG-3 is a class of immunosuppressive molecules and one of the co-receptor components of TCR. It intervenes in TCR activation of T lymphocytes and plays a negative regulatory function in T lymphocyte activation. In some diseases, LAG-3 expression will increase and corresponding immunosuppression will occur. Gandhi et al. found that LAG-3 was highly expressed in lymphocytes in the blood and tumor tissues of Hodgkin lymphoma patients; the function of specific CD8 ⁺ T cells in tumor tissues was significantly impaired. If LAG-3-positive T cells were removed, their anti-tumor function could be restored and cytokine secretion increased. Based on this, it is speculated that the expression of LAG-3 is related to the negative immune regulatory function of specific T cells. Inhibiting the function of LAG-3 molecules can enhance the anti-tumor effect of T cells. This molecule may be a potential target for tumor immunotherapy (Blood, 2006, 108(7): 2280-9).

Currently, many multinational pharmaceutical companies such as BMS and Novartis are developing monoclonal antibodies targeting LAG-3. They stimulate antigen-specific T cell responses and enhance the anti-tumor effect of T cells, thereby maximizing the patient's own immune system response to tumors and achieving the purpose of killing tumor cells.

However, due to their large molecular weight and complex structure, antibody drugs are easily degraded, aggregated, or undergo undesirable chemical modifications, making them unstable. In order to make antibodies suitable for drug administration and maintain stability during storage and subsequent use, and to exert better effects, the study of stable formulations of antibody drugs is particularly important.

Although, there are currently several companies developing LAG3 antibodies and their preparations, such as: WO2018204374, WO2010019570, WO2014008218, WO9530750, WO2004078928, WO2008132601, WO2014140180, WO2015138920, etc., there are not many studies on new LAG3 antibody preparations, so it is still necessary to develop a pharmaceutical composition (preparation) containing LAG3 that is more suitable for drug administration.

The present invention provides a pharmaceutical composition comprising a LAG3 antibody or an antigen-binding fragment thereof, and a buffer, wherein the buffer is selected from an acetate buffer, a histidine buffer, a citrate buffer, a succinate buffer or a Tris buffer.

In an optional embodiment, the acetate buffer in the pharmaceutical composition is selected from acetic acid-sodium acetate buffer, acetic acid-potassium acetate buffer, acetate-histidine buffer, acetic acid-calcium acetate buffer, acetic acid-magnesium acetate buffer, preferably acetic acid-sodium acetate buffer.

In an optional embodiment, the histidine buffer in the pharmaceutical composition is selected from histidine-hydrochloride buffer, histidine-acetate buffer, histidine-phosphate buffer, histidine-sulfate buffer, preferably histidine-hydrochloride buffer.

In an optional embodiment, the citrate buffer in the pharmaceutical composition is selected from citric acid-sodium citrate buffer; the succinate buffer is selected from succinic acid-sodium succinate buffer.

In an alternative embodiment, the concentration of the LAG-3 antibody or antigen-binding fragment thereof in the pharmaceutical composition is about 1 mg/ml to 90 mg/ml, preferably about 10 mg/ml to 90 mg/ml, preferably about 20 mg/ml to 90 mg/ml, preferably about 30 mg/ml to 90 mg/ml, preferably about 40 mg/ml to 90 mg/ml, preferably about 50 mg/ml to 90 mg/ml, preferably about 60 mg/ml to 90 mg/ml, preferably about 70 mg/ml to 90 mg/ml, preferably about 80mg/ml to 90mg/ml, preferably about 10mg/ml to 80mg/ml, preferably about 20mg/ml to 80mg/ml, preferably about 30mg/ml to 80mg/ml, preferably about 40mg/ml to 80mg/ml, preferably about 50mg/ml to 80mg/ml, preferably about 60mg/ml to 80mg/ml, preferably about 70mg/ml to 80mg/ml, preferably about 10mg/ml to 70mg/ml, preferably about About 20mg/ml to 70mg/ml, preferably about 30mg/ml to 70mg/ml, preferably about 40mg/ml to 70mg/ml, preferably about 50mg/ml to 70mg/ml, preferably about 60mg/ml to 70mg/ml, preferably about 10mg/ml to 60mg/ml, preferably about 20mg/ml to 60mg/ml, preferably about 30mg/ml to 60mg/ml, preferably about 40mg/ml to 60mg/ml, preferably about 50mg/ml To 60mg/ml, preferably about 10mg/ml to 50mg/ml, preferably about 20mg/ml to 50mg/ml, preferably about 30mg/ml to 50mg/ml, preferably about 40mg/ml to 50mg/ml, preferably about 10mg/ml to 40mg/ml, preferably about 20mg/ml to 40mg/ml, preferably about 30mg/ml to 40mg/ml, preferably about 10mg/ml to 30mg/ml, preferably about 20mg/ml to 30mg/ml. As a non-limiting example, the LAG-3 antibody or antigen-binding fragment thereof is present at a concentration of about 40 mg/ml, 41 mg/ml, 42 mg/ml, 43 mg/ml, 44 mg/ml, 45 mg/ml, 46 mg/ml, 47 mg/ml, 48 mg/ml, 49 mg/ml, 50 mg/ml, 51 mg/ml, 52 mg/ml, 53 mg/ml, 54 mg/ml, 55 mg/ml, 56 mg/ml, 57 mg/ml, 58 mg/ml, 59 mg/ml or 60 mg/ml, preferably 50 mg/ml.

In an alternative embodiment, the concentration of the buffer is about 5mM to 30mM, preferably about 10mM to 30mM, preferably about 15mM to 30mM, preferably about 20mM to 30mM, preferably about 25mM to 30mM, preferably about 5mM to 25mM, preferably about 10mM to 25mM, preferably about 15mM to 25mM M, preferably about 20mM to 25mM, preferably about 5mM to 20mM, preferably about 10mM to 15mM; non-limiting examples of the buffer concentration are about 10mM, 12mM, 14mM, 16mM, 18mM, 20mM, 22mM, 24mM, 26mM, 28mM or 30mM, with 10mM being the best.

In an alternative embodiment, the pH of the buffer in the pharmaceutical composition is about 5.0 to 7.5, preferably about 5.5 to 7.5, preferably about 6.0 to 7.5, preferably about 6.5 to 7.5, preferably about 7.0 to 7.5, preferably about 5.0 to 7.0, preferably about 5.5 to 7.0, preferably about 6.0 to 7.0, preferably about 6.5 to 7.0, preferably about 5.0 to 6.5, preferably about 5.5 to 6.5, preferably about 6.0 to 6.5, preferably about 5.0 to 6.0, preferably about 5.5 to 6.0, preferably about 5.0 to 5.5, non-limiting examples of the pH value of the buffer may also be about 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, more preferably about 5.5-6.0, more preferably 5.5 or 6.0.

Furthermore, in an optional embodiment, the pharmaceutical composition also includes an excipient, which is selected from one or more of sugars and surfactants.

In an alternative embodiment, the sugar is a disaccharide, preferably trehalose, sucrose, mannitol or sorbitol, and more preferably sucrose. In an alternative embodiment, the concentration of the sugar in the pharmaceutical composition is about 30 mg/ml to 90 mg/ml, preferably about 60 mg/ml to 90 mg/ml, 35 mg/ml to 90 mg/ml, preferably about 40 mg/ml to 90 mg/ml, preferably about 45 mg/ml to 90 mg/ml, preferably about 50 mg/ml to 90 mg/ml, preferably about 55 mg/ml to 90 mg/ml, preferably about 60 mg/ml to 90 mg/ml, preferably about 65 mg/ml to 90 mg/ml, preferably about 70 mg/ml to 90 mg/ml l, preferably about 75mg/ml to 90mg/ml, preferably about 80mg/ml to 90mg/ml, preferably about 85mg/ml to 90mg/ml, preferably about 30mg/ml to 85mg/ml, preferably about 35mg/ml to 85mg/ml, preferably about 40mg/ml to 85mg/ml, preferably about 45mg/ml to 85mg/ml, preferably about 50mg/ml to 85mg/ml, preferably about 55mg/ml to 85mg/ml, preferably about 60mg/ml to 85mg/ml, preferably about 65mg/ml To 85mg/ml, preferably about 70mg/ml to 85mg/ml, preferably about 75mg/ml to 85mg/ml, preferably about 80mg/ml to 85mg/ml, preferably about 30mg/ml to 80mg/ml, preferably about 35mg/ml to 80mg/ml, preferably about 40mg/ml to 80mg/ml, preferably about 45mg/ml to 80mg/ml, preferably about 50mg/ml to 80mg/ml, preferably about 55mg/ml to 80mg/ml, preferably about 60mg/ml to 80mg/ml, preferably about 65mg/ml to 80mg/ml, preferably about 70mg/ml to 80mg/ml, preferably about 75mg/ml to 80mg/ml, preferably about 30mg/ml to 75mg/ml, preferably about 35mg/ml to 75mg/ml, preferably about 40mg/ml to 75mg/ml, preferably about 45mg/ml to 75mg/ml, preferably about 50mg/ml to 75mg/ml, preferably about 55mg/ml to 75mg/ml, preferably about 60mg/ml to 75mg/ml, preferably about 65mg/ml to 75mg/ml l, preferably about 70mg/ml to 75mg/ml, preferably about 30mg/ml to 70mg/ml, preferably about 35mg/ml to 70mg/ml, preferably about 40mg/ml to 70mg/ml, preferably about 45mg/ml to 70mg/ml, preferably about 50mg/ml to 70mg/ml, preferably about 55mg/ml to 70mg/ml, preferably about 60mg/ml to 70mg/ml, preferably about 65mg/ml to 70mg/ml, preferably about 30mg/ml to 65mg/ml, preferably about 35mg/ml l to 65mg/ml, preferably about 40mg/ml to 65mg/ml, preferably about 45mg/ml to 65mg/ml, preferably about 50mg/ml to 65mg/ml, preferably about 55mg/ml to 65mg/ml, preferably about 60mg/ml to 65mg/ml, preferably about 30mg/ml to 60mg/ml, preferably about 35mg/ml to 60mg/ml, preferably about 40mg/ml to 60mg/ml, preferably about 45mg/ml to 60mg/ml, preferably about 50mg/ml to 60mg/ml, preferably about 55mg/ml to 60mg/ml, preferably about 30mg/ml to 55mg/ml, preferably about 35mg/ml to 55mg/ml, preferably about 40mg/ml to 55mg/ml, preferably about 45mg/ml to 55mg/ml, preferably about 50mg/ml to 55mg/ml, preferably about 30mg/ml to 50mg/ml, preferably about 35mg/ml to 50mg/ml, preferably about 40mg/ml to 50mg/ml, preferably about 45mg/ml to 50mg/ml, preferably about 30mg/ml to 45mg/ml ml, preferably about 35mg/ml to 45mg/ml, preferably about 40mg/ml to 45mg/ml, preferably about 30mg/ml to 40mg/ml, preferably about 35mg/ml to 40mg/ml, preferably about 30mg/ml to 35mg/ml; as a non-limiting example, the concentration of sugar in the pharmaceutical composition is about 60mg/ml, 65mg/ml, 70mg/ml, 75mg/ml, 80mg/ml, 85mg/ml or 90mg/ml, more preferably about 70mg/ml-80mg/ml, and most preferably 75mg/ml.

In an optional embodiment, the pharmaceutical composition further comprises a surfactant. The surfactant may be selected from polysorbate 20, polysorbate 80, polyhydroxyethane, Triton, sodium dodecyl sulfonate, sodium lauryl sulfonate, sodium octyl glucoside, lauryl-sulfobetaine, myristyl-sulfobetaine, linoleyl-sulfobetaine, stearyl-sulfobetaine, lauryl-sarcosine, myristyl-sarcosine, linoleyl-sarcosine, stearyl-sarcosine, linoleyl-betaine, myristyl-betaine, cetyl-betaine, Lauroylamidopropyl-betaine, cocamidopropyl-betaine, linoleamidopropyl-betaine, myristamidopropyl-betaine, palmitoylpropyl-betaine, isostearamidopropyl-betaine, myristamidopropyl-dimethylamine, palmitoylpropyl-dimethylamine, isostearamidopropyl-dimethylamine, methyl cocoyl sodium, methyl oleyl sodium taurate, polyethylene glycol, polypropylene glycol, copolymer of ethylene and propylene glycol, etc. The preferred surfactant is polysorbate 80 or polysorbate 20, more preferably polysorbate 80.

In an alternative embodiment, the concentration of the surfactant in the pharmaceutical composition is about 0.02 mg/ml to 0.8 mg/ml, preferably about 0.1 mg/ml to 0.8 mg/ml, preferably about 0.2 mg/ml to 0.8 mg/ml, preferably about 0.3 mg/ml to 0.8 mg/ml, preferably about 0.4 mg/ml to 0.8 mg/ml, preferably about 0.5 mg/ml to 0.8 mg/ml, preferably about 0.6 mg/ml to 0.8 mg/ml, preferably about 0.7 mg/ml to 0.8 mg/ml, preferably about 0.02 mg/ml to 0.7 mg/ml, preferably about 0.1 mg/ml to 0.7mg/ml, preferably about 0.2mg/ml to 0.7mg/ml, preferably about 0.3mg/ml to 0.7mg/ml, preferably about 0.4mg/ml to 0.7mg/ml, preferably about 0.5mg/ml to 0.7mg/ml, preferably about 0.6mg/ml to 0.7mg/ml, preferably about 0.02mg/ml to 0.6mg/ml, preferably about 0.1mg/ml to 0.6mg/ml, preferably about 0.2mg/ml to 0.6mg/ml, preferably about 0.3mg/ml to 0.6mg/ml, preferably about 0.4mg/ml to 0.6mg/ml, preferably about 0.5mg/ml to 0.7mg/ml g/ml to 0.6mg/ml, preferably about 0.02mg/ml to 0.5mg/ml, preferably about 0.1mg/ml to 0.5mg/ml, preferably about 0.2mg/ml to 0.5mg/ml, preferably about 0.3mg/ml to 0.5mg/ml, preferably about 0.4mg/ml to 0.5mg/ml, preferably about 0.02mg/ml to 0.4mg/ml, preferably about 0.1mg/ml to 0.4mg/ml, preferably about 0.2mg/ml to 0.4mg/ml, preferably about 0.3mg/ml to 0.4mg/ml, preferably about 0.02mg/ml to 0.3mg/ ml, preferably about 0.1mg/ml to 0.3mg/ml, preferably about 0.2mg/ml to 0.3mg/ml, preferably about 0.02mg/ml to 0.2mg/ml, preferably about 0.1mg/ml to 0.2mg/ml, preferably about 0.02mg/ml to 0.1mg/ml, as a non-limiting example, the concentration of the surfactant in the pharmaceutical composition is about 0.2mg/ml, 0.25mg/ml, 0.3mg/ml, 0.35mg/ml, 0.4mg/ml, 0.45mg/ml or 0.5mg/ml, more preferably about 0.3mg/ml-0.5mg/ml.

In one embodiment, the pharmaceutical composition comprises the following components i) or ii): i) (a) 1 mg/ml to 90 mg/ml of a LAG-3 antibody or an antigen-binding fragment thereof, (b) 5 mM to 30 mM acetic acid-sodium acetate buffer, pH about 5.0-6.5, (c) 30 mg/ml to 90 mg/ml of sucrose, and (d) 0.02 mg/ml to 1.5 mg/ml of dapoxetine. 0.8 mg/ml of polysorbate 80; preferably, the pharmaceutical composition comprises: (e) 40 mg/ml to 80 mg/ml of LAG-3 antibody or antigen-binding fragment thereof, (f) 10 mM to 30 mM acetic acid-sodium acetate buffer, pH about 5.2-5.8, (g) 70 mg/ml to 80 mg/ml of sucrose, and (h) 0.4 mg/ml to 0.5 m g/ml of polysorbate 80; or ii) (a) 1 mg/ml to 90 mg/ml of LAG-3 antibody or antigen-binding fragment thereof, (b) 5 mM to 30 mM histidine-hydrochloric acid buffer, pH about 5.0-6.5, (c) 30 mg/ml to 90 mg/ml of sucrose or trehalose, and (d) 0.05 mg/ml to 0.6 mg/ml of polysorbate 80, preferably, the pharmaceutical composition comprises: (e) 45 mg/ml to 60 mg/ml of LAG-3 antibody or its antigen-binding fragment, (f) 10 mM to 30 mM histidine-hydrochloric acid buffer, pH about 5.5-6.0, (g) 60 mg/ml to 90 mg/ml of sucrose, and (h) 0.2 mg/ml to 0.6 mg/ml of polysorbate 80.

In one embodiment, the LAG3 antibody or antigen-binding fragment thereof in the pharmaceutical composition comprises heavy chain HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11, respectively, and light chain LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17, respectively.

In an optional embodiment, the pharmaceutical composition comprises: (a) 1 mg/ml to 90 mg/ml of LAG-3 antibody or antigen-binding fragment thereof, (b) 5 mM to 30 mM acetate buffer, preferably at a pH of about 5.0 to 6.5, (c) 30 mg/ml to 90 mg/ml of sucrose, and (d) 0.02 mg/ml to 0.8 mg/ml of polysorbate 80.

In an optional embodiment, the pharmaceutical composition comprises: (a) 40 mg/ml to 80 mg/ml of LAG-3 antibody or antigen-binding fragment thereof, (b) 10 mM to 30 mM acetate buffer, pH about 5.0-6.0, (c) 60 mg/ml to 90 mg/ml of sucrose, and (d) 0.1 mg/ml to 0.5 mg/ml of polysorbate 80.

In an optional embodiment, the pharmaceutical composition comprises: (a) 50 mg/ml to 60 mg/ml of LAG-3 antibody or antigen-binding fragment thereof, (b) 10 mM to 30 mM acetic acid-sodium acetate buffer, and pH is about 5.2-5.8. (c) 70 mg to 80 mg/ml of sucrose, and (d) 0.4 mg/ml to 0.5 mg/ml of polysorbate 80.

In an alternative embodiment, the pharmaceutical composition comprises: (a) about 50 mg/ml of LAG-3 antibody or antigen-binding fragment thereof, (b) 10 mM acetic acid-sodium acetate buffer, pH about 5.5, (c) about 75 mg/ml of sucrose, and (d) about 0.4 mg/ml of polysorbate 80.

In an optional embodiment, the LAG-3 antibody or its antigen-binding fragment in the above-mentioned pharmaceutical composition is a murine antibody or its antigen-binding fragment, a chimeric antibody or its antigen-binding fragment, or a humanized antibody or its antigen-binding fragment.

In an optional embodiment, the LAG3 mouse antibody in the above-mentioned pharmaceutical composition comprises a heavy chain variable region as shown in SEQ ID NO: 5 and a light chain variable region as shown in SEQ ID NO: 6.

In an optional embodiment, the LAG-3 humanized antibody in the above pharmaceutical composition comprises a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is as shown in any one of SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 24 or SEQ ID NO: 25, or an amino acid sequence having at least 85% sequence identity therewith; wherein the light chain variable region is as shown in any one of SEQ ID NO: 22, SEQ ID NO: 26, SEQ ID NO: 27 or SEQ ID NO: 28, or an amino acid sequence having at least 85% sequence identity therewith.

In an alternative embodiment, the light chain variable region of the LAG-3 antibody in the pharmaceutical composition has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the light chain amino acid variable region sequence of SEQ ID NO: 22, SEQ ID NO: 26, SEQ ID NO: 27 or SEQ ID NO: 28, and the heavy chain amino acid variable region sequence of the LAG-3 antibody has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the antibody heavy chain variable region of SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 24 or SEQ ID NO: 25.

In an alternative embodiment, the LAG3 humanized antibody in the pharmaceutical composition comprises a combination of a heavy chain variable region and a light chain variable region selected from the following: 1) a heavy chain variable region of SEQ ID NO: 21 and a light chain variable region of SEQ ID NO: 22; 2) a heavy chain variable region of SEQ ID NO: 21 and a light chain variable region of SEQ ID NO: 26; 3) a heavy chain variable region of SEQ ID NO: 21 and a light chain variable region of SEQ ID NO: 27; 4) a heavy chain variable region of SEQ ID NO: 21 and a light chain variable region of SEQ ID NO: 28; 5) a heavy chain variable region of SEQ ID NO: 23 and a light chain variable region of SEQ ID NO: 22; 6) a heavy chain variable region of SEQ ID NO: 23 and a light chain variable region of SEQ ID NO: 26; 7) a heavy chain variable region of SEQ ID NO: 2 NO:23 heavy chain variable region and SEQ ID NO:27 light chain variable region; 8) SEQ ID NO:23 heavy chain variable region and SEQ ID NO:28 light chain variable region; 9) SEQ ID NO:24 heavy chain variable region and SEQ ID NO:22 light chain variable region; 10) SEQ ID NO:24 heavy chain variable region and SEQ ID NO:26 light chain variable region; 11) SEQ ID NO:24 heavy chain variable region and SEQ ID NO:27 light chain variable region; 12) SEQ ID NO:24 heavy chain variable region and SEQ ID NO:28 light chain variable region; 13) SEQ ID NO:25 heavy chain variable region and SEQ ID NO:22 light chain variable region; 14) SEQ ID NO:25 heavy chain variable region and SEQ ID NO:26 light chain variable region; NO: 26 light chain variable region; 15) SEQ ID NO: 25 heavy chain variable region and SEQ ID NO: 27 light chain variable region; and 16) SEQ ID NO: 25 heavy chain variable region and SEQ ID NO: 28 light chain variable region.

In an optional embodiment, the LAG-3 humanized antibody in the above pharmaceutical composition comprises a heavy chain constant region and a light chain constant region, wherein the heavy chain constant region is preferably as shown in SEQ ID NO: 38; wherein the light chain constant region is preferably as shown in SEQ ID NO: 39.

In an optional embodiment, the heavy chain of the LAG-3 humanized antibody in the pharmaceutical composition is selected from the sequence shown in SEQ ID NO: 40 or an amino acid sequence having at least 95% sequence identity therewith, and the light chain is selected from the sequence shown in SEQ ID NO: 41 or an amino acid sequence having at least 95% sequence identity therewith.

In an optional embodiment, the heavy chain of the LAG-3 antibody in the pharmaceutical composition has at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the heavy chain amino acid sequence shown in SEQ ID NO: 40, and the light chain amino acid sequence of the LAG-3 antibody has at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the antibody light chain shown in SEQ ID NO: 41.

In one embodiment, the pharmaceutical composition comprises: 50 mg/ml LAG3 antibody Hu229-013, 10 mM acetic acid-sodium acetate buffer, pH 5.5.

In another embodiment, the pharmaceutical composition comprises: 50 mg/ml of LAG3 antibody Hu229-013, 10 mM succinic acid-sodium succinate buffer, pH 6.0.

In another embodiment, the pharmaceutical composition comprises: 50 mg/ml LAG3 antibody Hu229-013, 10 mM histidine-hydrochloric acid buffer, pH 6.0.

In another embodiment, the pharmaceutical composition comprises: 50 mg/ml of LAG3 antibody Hu229-013, 10 mM succinate-sodium succinate, pH 6.0 and 0.1 mg/ml polysorbate 80.

In another embodiment, the pharmaceutical composition comprises: 50 mg/ml of LAG3 antibody Hu229-013, 10 mM succinate-sodium succinate, pH 6.0, and 70 mg/ml sucrose.

In another embodiment, the pharmaceutical composition comprises: 50 mg/ml of LAG3 antibody Hu229-013, 10 mM acetic acid-sodium acetate, pH 5.5, 60 mg/ml sucrose and 0.4 mg/mL polysorbate 80.

In another embodiment, the pharmaceutical composition comprises: 50 mg/ml of LAG3 antibody Hu229-013, 10 mM histidine-acetate, pH 6.0, 60 mg/ml sucrose and 0.4 mg/mL polysorbate 80.

In some embodiments, the pharmaceutical composition comprises: 1 mg/ml LAG3 antibody Hu229-013, 10-30 mM histidine-acetate, pH 5.5, 75 mg/ml sucrose and 0.2 mg/mL polysorbate 80.

In some other embodiments, the pharmaceutical composition comprises: 50 mg/ml of LAG3 antibody Hu229-013, 10-30 mM acetic acid-sodium acetate, pH 5.2-5.8, 75 mg/ml sucrose and 0.2 mg/mL polysorbate 80.

In another embodiment, the pharmaceutical composition comprises: 60 mg/ml of LAG3 antibody Hu229-013, 10 mM acetic acid-sodium acetate, pH 5.5, 60 mg/ml sucrose and 0.4 mg/mL polysorbate 80.

In some other embodiments, the pharmaceutical composition comprises: 50-60 mg/ml of LAG3 antibody Hu229-013, 10 mM acetic acid-sodium acetate, pH 5.5, 30-90 mg/ml sucrose and 0.4-0.5 mg/mL polysorbate 80.

In another embodiment, the pharmaceutical composition comprises: 50 mg/ml of LAG3 antibody Hu229-013, 10 mM acetic acid-sodium acetate, pH 5.5, 75 mg/ml sucrose and 0.4 mg/mL polysorbate 80.

In other embodiments, the LAG-3 antibody or antigen-binding fragment thereof in the pharmaceutical composition comprises HCDR1, HCDR2 and HCDR3 regions as shown in SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, respectively, and LCDR1, LCDR2 and LCDR3 regions as shown in SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20, respectively.

In an alternative embodiment, the pharmaceutical composition comprises: (a) 1 mg/ml to 90 mg/ml of LAG-3 antibody or antigen-binding fragment thereof, (b) 5 mM to 30 mM histidine-hydrochloric acid buffer, pH about 5.0-6.5, (c) 30 mg/ml to 90 mg/ml of sucrose or trehalose, and (d) 0.05 mg/ml to 0.6 mg/ml of polysorbate 80.

In an alternative embodiment, the pharmaceutical composition comprises: (a) 45 mg/ml to 60 mg/ml of LAG-3 antibody or antigen-binding fragment thereof, (b) about 10 mM to 30 mM of histidine-sodium hydrochloride buffer, pH about 5.0-6.0, (c) 60 mg to 90 mg/ml of sucrose or trehalose, and (d) 0.2 mg/ml to 0.6 mg/ml of polysorbate 80.

In an optional embodiment, the pharmaceutical composition comprises: (a) about 50 mg/ml of LAG-3 antibody or antigen-binding fragment thereof, (b) 10 mM histidine-hydrochloric acid buffer, pH about 5.0, (c) about 75 mg/ml of sucrose or trehalose, and (d) about 0.3 mg/ml of polysorbate 80.

In an optional embodiment, the LAG-3 antibody or its antigen-binding fragment in the above-mentioned pharmaceutical composition is a murine antibody or its fragment, a chimeric antibody or its antigen-binding fragment, or a humanized antibody or its antigen-binding fragment.

In an optional embodiment, the LAG3 mouse antibody in the above-mentioned pharmaceutical composition comprises a heavy chain variable region as shown in SEQ ID NO: 7 and a light chain variable region as shown in SEQ ID NO: 8.

In an optional embodiment, the LAG-3 humanized antibody in the above pharmaceutical composition comprises: A heavy chain variable region sequence selected from any one of SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 32 or SEQ ID NO: 33 or an amino acid sequence having at least 85% sequence identity therewith and a light chain variable region sequence selected from any one of SEQ ID NO: 30, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36 or SEQ ID NO: 37 or an amino acid sequence having at least 85% sequence identity therewith.

In alternative embodiments, the light chain variable region of the LAG-3 antibody in the pharmaceutical composition has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the light chain amino acid variable region sequence of SEQ ID NO: 30, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36 or SEQ ID 37, and the heavy chain amino acid variable region sequence of the LAG-3 antibody has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the light chain amino acid variable region sequence of SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 32 or SEQ ID The antibody heavy chain variable region shown in NO: 33 has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

In an optional embodiment, the LAG3 humanized antibody in the pharmaceutical composition comprises a combination of a heavy chain variable region and a light chain variable region selected from the following: 1) a heavy chain variable region of SEQ ID NO: 29 and a light chain variable region of SEQ ID NO: 30; 2) a heavy chain variable region of SEQ ID NO: 29 and a light chain variable region of SEQ ID NO: 34; 3) a heavy chain variable region of SEQ ID NO: 29 and a light chain variable region of SEQ ID NO: 35; 4) a heavy chain variable region of SEQ ID NO: 29 and a light chain variable region of SEQ ID NO: 36; 5) a heavy chain variable region of SEQ ID NO: 29 and a light chain variable region of SEQ ID NO: 37; 6) a heavy chain variable region of SEQ ID NO: 31 and a light chain variable region of SEQ ID NO: 30; 7) a heavy chain variable region of SEQ ID NO: 29 and a light chain variable region of SEQ ID NO: 35; NO:31 and the light chain variable region of SEQ ID NO:34; 8) the heavy chain variable region of SEQ ID NO:31 and the light chain variable region of SEQ ID NO:35; 9) the heavy chain variable region of SEQ ID NO:31 and the light chain variable region of SEQ ID NO:36; 10) the heavy chain variable region of SEQ ID NO:31 and the light chain variable region of SEQ ID NO:37; 11) the heavy chain variable region of SEQ ID NO:32 and the light chain variable region of SEQ ID NO:30; 12) the heavy chain variable region of SEQ ID NO:32 and the light chain variable region of SEQ ID NO:34; 13) the heavy chain variable region of SEQ ID NO:32 and the light chain variable region of SEQ ID NO:35; 14) the heavy chain variable region of SEQ ID NO:32 and the light chain variable region of SEQ ID NO: NO:36 light chain variable region; 15) SEQ ID NO:32 heavy chain variable region and SEQ ID NO:37 light chain variable region; 16) SEQ ID NO:33 heavy chain variable region and SEQ ID NO:30 light chain variable region; 17) SEQ ID NO:33 heavy chain variable region and SEQ ID NO:34 light chain variable region; 18) SEQ ID NO:33 heavy chain variable region and SEQ ID NO:35 light chain variable region; 19) SEQ ID NO:33 heavy chain variable region and SEQ ID NO:36 light chain variable region; and 20) SEQ ID NO:33 heavy chain variable region and SEQ ID NO:37 light chain variable region.

In an optional embodiment, the chimeric antibody or humanized antibody heavy chain in the above pharmaceutical composition further comprises a heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4 or a variant thereof, preferably a heavy chain constant region of human IgG4 or a variant thereof, and most preferably a heavy chain constant region as shown in SEQ ID NO: 38; the chimeric antibody or humanized antibody light chain further comprises a light chain constant region of human κ, λ chain or a variant thereof, preferably a light chain constant region as shown in SEQ ID NO: 39.

In an optional embodiment, the heavy chain of the LAG-3 humanized antibody in the above pharmaceutical composition is selected from the sequence shown in SEQ ID NO: 42 or an amino acid sequence having at least 95% sequence identity therewith, and the light chain is selected from the sequence shown in SEQ ID NO: 43 or an amino acid sequence having at least 95% sequence identity therewith.

In an optional embodiment, the heavy chain of the LAG-3 antibody in the pharmaceutical composition has at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the heavy chain amino acid sequence shown in SEQ ID NO: 42, and the light chain amino acid sequence of the LAG-3 antibody has at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the antibody light chain shown in SEQ ID NO: 43.

In one embodiment, the pharmaceutical composition comprises: 50 mg/ml LAG3 antibody Hu303-005, 10 mM histidine-hydrochloric acid buffer, pH 6.0.

In one embodiment, the pharmaceutical composition comprises: 50 mg/ml LAG3 antibody Hu303-005, 10 mM histidine-hydrochloric acid buffer, pH 5.0.

In one embodiment, the pharmaceutical composition comprises: 50 mg/ml LAG3 antibody Hu303-005, 10 mM histidine-hydrochloric acid buffer, pH 6.5.

In another embodiment, the pharmaceutical composition comprises: 50 mg/ml LAG3 antibody Hu303-005, 10 mM acetic acid-sodium acetate buffer, pH 5.5.

In another embodiment, the pharmaceutical composition comprises: 50 mg/ml of LAG3 antibody Hu303-005, 10 mM acetic acid-sodium acetate buffer, pH 5.5, 75 mg/ml sucrose and 0.2 mg/ml polysorbate 80.

In another embodiment, the pharmaceutical composition comprises: 50 mg/ml of LAG3 antibody Hu303-005, 10 mM acetic acid-sodium acetate buffer, pH 5.5, 75 mg/ml trehalose and 0.2 mg/ml polysorbate 80.

In another embodiment, the pharmaceutical composition comprises: 50 mg/ml of LAG3 antibody Hu303-005, 10 mM acetic acid-sodium acetate buffer, pH 5.5, and 0.4 mg/ml polysorbate 80.

In another embodiment, the pharmaceutical composition comprises: 50 mg/ml of LAG3 antibody Hu303-005, 10 mM histidine-hydrochloric acid buffer, pH 6.0, 75 mg/ml sucrose and 0.4 mg/ml polysorbate 80.

In another embodiment, the pharmaceutical composition comprises: 50 mg/ml of LAG3 antibody Hu303-005, 10 mM histidine-hydrochloric acid buffer, pH 6.5, 75 mg/ml sucrose and 0.4 mg/ml polysorbate 80.

In another embodiment, the pharmaceutical composition comprises: 50 mg/ml of LAG3 antibody Hu303-005, 10 mM histidine-hydrochloric acid buffer, pH 5.5, 75 mg/ml sucrose and 0.4 mg/ml polysorbate 80.

In some embodiments, the pharmaceutical composition comprises: 45-60 mg/ml of LAG3 antibody Hu303-005, 10 mM histidine-hydrochloric acid buffer, pH 5.5, 75 mg/ml sucrose and 0.2-0.6 mg/ml polysorbate 80.

In another embodiment, the pharmaceutical composition comprises: about 50 mg/ml of LAG3 antibody Hu303-005, about 10 mM histidine-hydrochloric acid buffer, about pH 6.0, about 75 mg/ml sucrose and about 0.3 mg/ml polysorbate 80.

The present invention also provides a method for preparing a pharmaceutical composition containing a LAG-3 antibody, comprising mixing a LAG-3 antibody or an antigen-binding fragment with a pharmaceutically acceptable excipient.

The present invention also provides a method for preparing a freeze-dried preparation containing LAG-3 antibodies, which includes a step of freeze-drying the aforementioned pharmaceutical composition.

In an optional embodiment, the freeze-drying in the method for preparing a freeze-dried preparation containing LAG-3 antibody includes the steps of pre-freezing, primary drying and secondary drying in sequence.

In an optional embodiment, the primary drying temperature in the method for preparing a freeze-dried preparation containing LAG-3 antibodies is -5°C to -20°C, preferably -10°C.

The present invention also provides a lyophilized preparation containing LAG-3 antibodies prepared by the aforementioned method for preparing a lyophilized preparation containing LAG-3 antibodies.

In some embodiments, the lyophilized formulation is stable at 2-8°C for at least 3 months, at least 6 months, at least 12 months, at least 18 months, or at least 24 months. In some embodiments, the lyophilized formulation is stable at 40°C for at least 7 days, at least 14 days, or at least 28 days.

The present invention also provides a lyophilized preparation containing LAG-3 antibody prepared by the above-mentioned lyophilization method.

The present invention also provides a freeze-dried preparation containing LAG3 antibodies, which is characterized in that the preparation can form the above-mentioned pharmaceutical composition after reconstitution.

The present invention also provides a method for preparing a reconstituted solution of a lyophilized preparation containing a LAG-3 antibody, which includes a step of reconstituted the aforementioned lyophilized preparation, and the solution used for reconstitution is selected from but not limited to water for injection, physiological saline or glucose solution.

The present invention also provides a method for preparing a reconstituted solution of a lyophilized preparation containing a LAG-3 antibody by the aforementioned method of reconstituted solution of a lyophilized preparation containing a LAG-3 antibody.

The present invention also provides a reconstituted solution containing LAG3 antibody, which also contains the following components: (a) 1 to 90 mg/ml of LAG-3 antibody or its antigen-binding fragment, (b) 5 to 30 mM acetic acid-sodium acetate buffer, pH about 5.0-6.5, (c) 30 to 90 mg/ml of sucrose, (d) 0.02 to 0.8 mg/ml of polysorbate 80.

The present invention also provides a reconstituted solution containing LAG3 antibody, which also contains the following components: (a) 40 to 80 mg/ml of LAG-3 antibody or its antigen-binding fragment, (b) 10 to 30 mM acetic acid-sodium acetate buffer, pH about 5.2-5.8, (c) 70 to 80 mg/ml sucrose, (d) 0.4 to 0.5 mg/ml polysorbate 80.

The present invention also provides a reconstituted solution containing LAG3 antibody, which also contains the following components: (a) 50 mg/ml LAG-3 antibody or its antigen-binding fragment, (b) 10 mM acetic acid-sodium acetate buffer, pH about 5.5, (c) 75 mg/ml sucrose, (d) 0.4 mg/ml polysorbate 80.

(a) 1 to 90 mg/ml of LAG-3 antibody or its antigen-binding fragment, (b) 5 to 30 mM of histidine-hydrochloric acid buffer, pH about 5.0-6.5, (c) 30 to 90 mg/ml of sucrose, (d) 0.05 to 0.6 mg/ml of polysorbate 80.

The present invention also provides a reconstituted solution containing LAG3 antibody, which also contains the following components: (a) 45 to 60 mg/ml of LAG-3 antibody or its antigen-binding fragment, (b) 10 to 30 mM histidine-hydrochloric acid buffer, pH about 5.5-6.0, (c) 60 to 80 mg/ml of sucrose, (d) 0.2 to 0.6 mg/ml of polysorbate 80.

The present invention also provides a reconstituted solution containing LAG3 antibody, which also contains the following components: (a) 50 mg/ml LAG-3 antibody or its antigen-binding fragment, (b) 10 mM histidine-hydrochloric acid buffer, pH about 6.0, (c) 75 mg/ml sucrose, (d) 0.3 mg/ml polysorbate 80.

The present invention further provides a product or test kit comprising a container containing any stable pharmaceutical composition described herein. In some embodiments, the glass bottle is a neutral borosilicate glass tube injection bottle.

The aforementioned pharmaceutical composition or lyophilized preparation or reconstituted solution of lyophilized preparation of the present invention can be used as a medicine.

The present invention also provides the use of the aforementioned pharmaceutical composition or lyophilized preparation or reconstituted solution of the lyophilized preparation in the preparation of a drug for treating a LAG-3-related disease or condition, wherein the disease or condition is a disease or condition involving pathogenic T cells, preferably cancer; the cancer includes, but is not limited to, ovarian cancer, melanoma, prostate cancer, intestinal cancer, gastric cancer, esophageal cancer, breast cancer, lung cancer, kidney cancer, pancreatic cancer, uterine cancer, liver cancer, bladder cancer, cervical cancer, oral cancer, brain cancer, testicular cancer, skin cancer, thyroid cancer and hematological malignancies, wherein the hematological malignancies include myeloma and chronic and acute leukemia.

The present invention also provides a method for treating and preventing LAG-3-related diseases or conditions, comprising administering a therapeutically effective amount of the aforementioned pharmaceutical composition or lyophilized preparation or a reconstituted solution of the lyophilized preparation to a patient in need thereof, wherein the disease or condition is a disease or condition related to pathogenic T cells, preferably cancer; the cancer includes, but is not limited to, ovarian cancer, melanoma, prostate cancer, intestinal cancer, gastric cancer, esophageal cancer, breast cancer, lung cancer, kidney cancer, pancreatic cancer, uterine cancer, liver cancer, bladder cancer, cervical cancer, oral cancer, brain cancer, testicular cancer, skin cancer, thyroid cancer, and hematological malignancies, wherein the hematological malignancies include myeloma and chronic and acute leukemia.

The present invention also provides a product, which includes a container containing the aforementioned pharmaceutical composition or lyophilized preparation or a reconstituted solution of the lyophilized preparation.

As is well known to those skilled in the art, one, some or all of the features of each embodiment in this disclosure can be further combined to form other embodiments of the present invention. The above embodiments of the present invention and other embodiments obtained by combination are further described in detail below.

(Term)

In order to make the present invention easier to understand, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by ordinary technicians in the field to which the present invention belongs.

"Buffer" refers to a buffer that tolerates changes in pH by virtue of its acid-base conjugate component. Examples of buffers that control pH within an appropriate range include acetate, succinate, gluconate, histidine, oxalate, lactate, phosphate, citrate, tartrate, fumarate, glycine, and other organic acid buffers.

"Histidine buffer" is a buffer containing histidine ions. Examples of histidine buffers include histidine hydrochloride, histidine acetate, histidine phosphate, histidine sulfate and the like, preferably histidine hydrochloride buffer. Histidine hydrochloride buffer is prepared by histidine and hydrochloric acid or histidine and histidine hydrochloride.

"Citrate buffer" is a buffer including citrate ions. Examples of citrate buffers include citric acid-sodium citrate, citric acid-potassium citrate, citric acid-calcium citrate, citric acid-magnesium citrate, etc. A preferred citrate buffer is citric acid-sodium citrate buffer.

"Succinate buffer" is a buffer including succinic acid ions. Examples of succinate buffers include succinate-sodium succinate, succinate-potassium succinate, succinate-calcium succinate, etc. A preferred succinate buffer is succinate-sodium succinate buffer.

"Phosphate buffer" is a buffer including phosphate ions. Examples of phosphate buffers include sodium dihydrogen phosphate-sodium dihydrogen phosphate, sodium dihydrogen phosphate-potassium dihydrogen phosphate, etc. A preferred phosphate buffer is sodium dihydrogen phosphate-sodium dihydrogen phosphate buffer.

"Acetate buffer" is a buffer including acetate ions. Examples of acetate buffers include acetic acid-sodium acetate, acetic acid-histidine salt, acetic acid-potassium acetate, acetic acid-calcium acetate, acetic acid-magnesium acetate, etc. A preferred acetate buffer is acetic acid-sodium acetate buffer.

"Tris buffer" refers to a buffer solution containing tris(hydroxymethyl)aminomethane. Tris buffer is also known as Tris base, Trizma, Trisamine, THAM, tromethamine, tromethamine and trisamine. The effective buffering range of Tris buffer is between pH 7.0 and 9.2. The pH of the aqueous solution of Tris base is about 10.5. Generally, hydrochloric acid is added to adjust the pH value to the required value to obtain a buffer of that pH value.

The "sugar" of the present invention includes conventional compositions ( _CH2O ) _n and derivatives thereof, including monosaccharides, disaccharides, trisaccharides, polysaccharides, sugar alcohols, reducing sugars, non-reducing sugars, etc. It can be selected from glucose, sucrose, trehalose, lactose, fructose, maltose, dextran, glycerol, erythritol, glycerol, arabitol, sylitol, sorbitol, mannitol, melibiose, melezitose, raffinose, mannotriose, stachyose, maltose, lactulose, maltulose, sorbitol, maltitol, lactitol, isomaltulose, etc. Preferred sugars are non-reducing disaccharides, and more preferably sucrose.

"Viscosity regulator" is a conventional pharmaceutical material added to adjust the viscosity of the preparation. The viscosity regulator referred to here mainly refers to inorganic salts and amino acid salts, wherein the inorganic salt is preferably selected from sodium chloride, calcium chloride, magnesium chloride, and calcium acetate, and the amino acid salt is preferably selected from arginine hydrochloride, histidine hydrochloride, glycine hydrochloride, histidine acetate, etc.

"Pharmaceutical composition" means a mixture containing one or more compounds or their physiologically/pharmaceutically acceptable salts or prodrugs and other chemical components, such as physiologically/pharmaceutically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to an organism, facilitate the absorption of the active ingredient, and thus exert biological activity. In this article, "pharmaceutical composition" and "preparation" are not mutually exclusive.

The solution form of the pharmaceutical composition described in the present invention, unless otherwise specified, contains water as the solvent.

"Lyophilized preparation" means a pharmaceutical composition in liquid or solution form or a preparation or pharmaceutical composition obtained after a solution preparation has been subjected to a vacuum freeze-drying step.

The freeze drying disclosed in the present invention includes pre-freezing, primary drying, and secondary drying. The purpose of pre-freezing is to freeze the product and obtain a crystalline solid. The pre-freezing temperature and the pre-freezing speed are two important process parameters. In the present invention, the pre-freezing temperature is set to -45°C and the pre-freezing speed is set to 1°C/min. Primary drying is also called main drying, which is the main stage of sample freeze drying. The purpose is to remove the ice in the product while maintaining the shape of the product and minimizing the damage to the product. If the temperature and vacuum degree of primary drying are not properly selected, it will cause the product to collapse. Higher temperature and vacuum degree will accelerate the freeze drying efficiency, but at the same time will increase the risk of product collapse. The primary drying temperature of the present invention can be a conventional temperature in the art, such as -30℃-0℃. Secondary drying, also known as analytical drying, is the main step of removing bound water in the product by drawing an extreme vacuum (0.01mbar) and raising the temperature (20-40℃). Since most biological products are sensitive to temperature, the secondary drying temperature is selected at the lower point of the temperature range, i.e. 25℃. The freeze drying time is related to the freezer, the dosage of the freeze-dried preparation, and the container of the freeze-dried medicine. The adjustment of this time is well known to technicians in this field.

The term "about" as used herein means that a numerical value is within an acceptable error range of a specific value determined by a person of ordinary skill in the art, which value depends in part on how it is measured or determined (i.e., the limits of the measurement system). For example, "about" can mean a standard deviation within or exceeding 1 in each practice in the art. Alternatively, "about" or "substantially including" can mean a range of up to 20%, for example, a pH of about 5.5 means pH 5.5±1.1. In addition, particularly for biological systems or processes, the term can mean up to an order of magnitude or up to 5 times the value. Unless otherwise stated, when a specific value appears in this application and the scope of the patent application, the meaning of "about" or "substantially including" should be assumed to be within an acceptable error range for the specific value.

The pharmaceutical composition of the present invention can achieve a stabilizing effect: the antibody therein substantially retains its physical stability and/or chemical stability and/or biological activity after storage. Preferably, the pharmaceutical composition substantially retains its physical and chemical stability and its biological activity after storage. The storage period is generally selected based on the predetermined shelf life of the pharmaceutical composition. There are currently a variety of analytical techniques for measuring protein stability, which can measure stability after storage at a selected temperature for a selected period of time.

A stable drug antibody formulation is one in which no significant change is observed when stored at refrigerated temperature (2-8°C) for at least 3 months, preferably 6 months, more preferably 1 year, and even more preferably up to 2 years. In addition, a stable liquid formulation includes a liquid formulation that exhibits the desired characteristics after a period of storage including 1 month, 3 months, 6 months at 25°C or 1 month at 40°C. Typical acceptable standards for stability are as follows: typically no more than about 5%, preferably no more than about 5%, of the antibody monomers are degraded as measured by SEC-HPLC. By visual analysis, the drug antibody formulation is colorless, or clear to slightly opalescent. The concentration, pH and osmotic pressure concentration of the formulation have a variation of no more than ±5%. Typically no more than about 5%, preferably no more than about 5%, truncation is observed, and typically no more than about 5%, preferably no more than about 5%, aggregation is formed.

An antibody "retains its physical stability" in a pharmaceutical formulation if it shows no significant increase in aggregation, precipitation, and/or denaturation after visual inspection of color and/or clarity, or as measured by UV light scattering, size exclusion chromatography (SEC), and dynamic light scattering (DLS). Changes in protein conformation can be assessed by fluorescence spectroscopy, which determines protein tertiary structure, and by FTIR spectroscopy, which determines protein secondary structure.

An antibody "retains its chemical stability" in a pharmaceutical formulation if it shows no significant chemical changes. Chemical stability can be assessed by detecting and quantifying chemically altered forms of the protein. Degradation processes that often alter the chemical structure of a protein include hydrolysis or truncation (assessed by methods such as size exclusion chromatography and SDS-PAGE), oxidation (assessed by methods such as peptide chromatography coupled to mass spectrometry or MALDI/TOF/MS), deamination (assessed by methods such as ion exchange chromatography, capillary isoelectric focusing, peptide chromatography, isoaspartate measurement), and isomerization (assessed by measuring isoaspartate content, peptide chromatography, etc.).

An antibody "retains its biological activity" in a pharmaceutical formulation if its biological activity at a given time is within a predetermined range of the biological activity exhibited when the pharmaceutical formulation is prepared. The biological activity of an antibody can be determined, for example, by an antigen binding assay.

The term "LAG-3" refers to lymphocyte activation gene 3. The term "LAG-3" includes variants, isoforms, homologs, orthologs, and paralogs. The term "human LAG-3" refers to the complete amino acid sequence of human sequence LAG-3, such as human LAG-3 with Uniprot No.: P18627. LAG-3 is also known in the art, such as CD215. The human LAG-3 sequence may differ from the human LAG-3 with Uniprot No.: P18627 in having, for example, conservative mutations or mutations in non-conserved regions, and LAG-3 has substantially the same biological function as the human LAG-3 with Uniprot No.: P18627. For example, the biological function of human LAG-3 is to have an epitope in the extracellular domain of LAG-3, which is specifically bound by the antibody disclosed herein, or the biological function of human LAG-3 is to bind to MHC class II molecules.

A specific human LAG-3 sequence is typically at least 90% identical in amino acid sequence to human LAG-3 of Uniprot No.: P18627, and contains amino acid residues that are identified as human amino acid sequences when compared to LAG-3 amino acid sequences of other species (e.g., mice). In certain instances, human LAG-3 may be at least 85% or even at least 95%, 96%, 97%, 98%, or 99% identical in amino acid sequence to LAG-3 of Uniprot No.: P18627. In certain embodiments, the human LAG-3 sequence exhibits no more than 10 amino acid differences from the LAG-3 sequence of Uniprot No.: P18627. In certain embodiments, human LAG-3 may exhibit no more than 5, or even no more than 4, 3, 2, or 1 amino acid differences from the LAG-3 sequence of Uniprot No.: P18627. The percent identity may be determined as described herein.

The three-letter codes and single-letter codes of amino acids used in the present invention are as described in J.biol.chem, 243, p3558 (1968).

The "antibody" mentioned in the present invention refers to immunoglobulin, which is a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains connected by interchain disulfide bonds.

In the present invention, the antibody light chain described in the present invention may further comprise a light chain constant region, and the light chain constant region comprises a human or mouse κ, λ chain or a variant thereof.

In the present invention, the antibody heavy chain described in the present invention may further comprise a heavy chain constant region, and the heavy chain constant region comprises human or mouse IgG1, IgG2, IgG3, IgG4 or a variant thereof.

The sequences of about 15 amino acids near the N-terminus of the antibody heavy chain and light chain vary greatly, which is the variable region (Fv region); the remaining amino acid sequences near the C-terminus are relatively stable, which is the constant region. The variable region includes 3 hypervariable regions (HVR) and 4 relatively conserved framework regions (FR). The 3 hypervariable regions determine the specificity of the antibody, also known as the complementarity determining region (CDR). Each light chain variable region (LCVR) and heavy chain variable region (HCVR) consists of 3 CDR regions and 4 FR regions, and the order from the amino terminal to the carboxyl terminal is: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDR regions of the light chain are LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain are HCDR1, HCDR2, and HCDR3.

The antibodies of the present invention include mouse antibodies, chimeric antibodies, and humanized antibodies, preferably humanized antibodies.

The term "mouse antibody" in the present invention refers to a monoclonal antibody against human LAG-3 prepared according to the knowledge and skills in the art. During preparation, the test subject is injected with LAG-3 antigen, and then the fusion tumor expressing the antibody with the desired sequence or functional properties is isolated.

The term "chimeric antibody" refers to an antibody formed by fusing the variable region of a mouse antibody with the constant region of a human antibody, which can reduce the immune response induced by the mouse antibody. To establish a chimeric antibody, a fusion tumor that secretes mouse-specific monoclonal antibodies must first be established, and then variable region genes are selected from the mouse fusion tumor cells, and then the constant region genes of human antibodies are selected as needed. The mouse variable region genes and the human constant region genes are connected to form a chimeric gene and inserted into a human vector, and finally the chimeric antibody molecule is expressed in a eukaryotic or prokaryotic industrial system. In a preferred embodiment of the present invention, the antibody light chain of the LAG-3 chimeric antibody further comprises a light chain constant region of a human κ, λ chain or a variant thereof. The antibody heavy chain of the LAG-3 chimeric antibody further comprises a heavy chain constant region of human IgG1, IgG2, IgG3, IgG4 or a variant thereof.

The term "humanized antibody", also known as CDR-grafted antibody, refers to an antibody produced by transplanting mouse CDR sequences into human antibody variable region frameworks, i.e., different types of human germline antibody framework sequences. The strong antibody variable antibody reaction induced by chimeric antibodies carrying a large amount of mouse protein components can be overcome. Such framework sequences can be obtained from public DNA databases including germline antibody gene sequences or public references. For example, the germline DNA sequences of human heavy chain and light chain variable region genes can be found in the "VBase" human germline sequence database (available on the Internet at www.mrccpe.com.ac.uk/vbase), and in Kabat, E.A. et al., 1991 Sequences of Proteins of Immunological Interest, 5th edition. In order to avoid a decrease in activity caused by a decrease in immunogenicity, the variable region framework sequence of the human antibody can be subjected to a minimum reverse mutation or reversion mutation to maintain activity. The humanized antibodies of the present invention also include humanized antibodies after further affinity maturation of CDR by phage display.

The terms "anti-LAG-3 antibody", "anti-LAG-3", "LAG-3 antibody" or "antibody that binds to LAG-3" in the present invention refer to an antibody that is capable of binding to a LAG-3 antibody with sufficient affinity so that the antibody can be used as a diagnostic and/or therapeutic agent in targeting LAG-3.

The term "binding to LAG-3" in the present invention means being able to interact with human LAG-3.

The term "specific binding" refers to as determined by techniques available in the art, such as competitive ELISA, ^BIACORE® assay or ^KINEXA® assay. The term also applies when, for example, the antigen binding domain of an antibody of the invention is specific for a particular epitope carried by a number of antigens, in which case the antibody carrying the antigen binding domain is able to specifically bind to a plurality of antigens carrying that epitope.

The term "competitive binding" refers to an antibody that recognizes the same epitope (also called antigenic determinant) or a portion of the same epitope on the extracellular region of human LAG-3 as the monoclonal antibody of the present invention and binds to the antigen. An antibody that binds to the same epitope as the monoclonal antibody of the present invention refers to an antibody that recognizes and binds to the amino acid sequence of human LAG-3 recognized by the monoclonal antibody of the present invention.

The term "KD" or "Kd" refers to the dissociation equilibrium constant for a particular antibody-antigen interaction. Typically, an antibody of the invention binds to LAG-3 with a dissociation equilibrium constant (KD) of less than about ^10-7 M, e.g., less than about ^10-8 M, ^10-9 M, or ^10-10 M or less, e.g., as determined using surface plasmon resonance (SPR) technology in a BIACORE instrument.

The "antigen-binding fragment" described in the present invention refers to Fab fragments, Fab' fragments, F(ab')2 fragments, scFv fragments that bind to human LAG-3, and other fragments that can bind to human LAG-3 using the VH and VL regions of antibodies that can bind to human LAG-3; they contain one or more CDR regions selected from SEQ ID NOs: 9, 10, 11, 15, 16, 17 and 12, 13, 14, 18, 19 and 20 of the antibody described in the present invention. The Fv fragment contains the variable regions of the heavy chain and the variable regions of the light chain of the antibody, but does not have a constant region, and is the smallest antibody fragment that has all the antigen-binding sites. Generally, the Fv antibody also contains a polypeptide linker between the VH and VL domains, and is capable of forming the structure required for antigen binding. Different linkers can also be used to connect two antibody variable regions into a polypeptide chain, called a single chain antibody or single chain Fv (sFv).

The term "epitope" refers to the part of a molecule that can be recognized and bound by one or more antibodies in the antigen binding region of the antibody.

"Conservative modification" or "conservative replacement or substitution" refers to replacing amino acids in proteins with other amino acids with similar characteristics (such as charge, side chain size, hydrophobicity/hydrophilicity, main chain conformation and rigidity, etc.), so that changes can be made frequently without changing the biological activity of the protein. Those skilled in the art know that, in general, single amino acid replacement in non-essential regions of polypeptides does not substantially change biological activity (see, for example, Watson et al. (1987) Molecular Biology of the Gene, The Benjamin/Cummings Pub. Co., p. 224, (4th edition)). In addition, replacement of amino acids with similar structures or functions is unlikely to destroy biological activity.

Amino acid sequence "identity" refers to the sequence similarity between two proteins or polypeptides. When a position in both compared sequences is occupied by the same amino acid residue, for example, if a position in both polypeptides is occupied by the same amino acid residue, then the molecules are identical at that position. Examples of algorithms suitable for determining percent sequence identity and percent sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1990) J. Mol. Biol. 215: 403-45 and Altschul et al. (1977) Nucleic Acids Res. 25: 3389-3402, respectively. Software for performing BLAST analyses is publicly available at the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/).

Methods for producing and purifying antibodies and antigen-binding fragments are well known in the prior art, such as the Cold Spring Harbor Guide to Antibody Experimental Techniques, Chapters 5-8 and 15. The antibody or antigen-binding fragment of the invention uses genetic engineering methods to add one or more human FR regions to the non-human CDR region. Human FR germline sequences can be obtained from the ImMunoGeneTics (IMGT) website http://imgt.cines.fr by aligning the IMGT human antibody variable region germline gene database and MOE software, or from the Immunoglobulin Journal, 2001 ISBN012441351.

The engineered antibodies or antigen-binding fragments of the present invention can be prepared and purified by conventional methods. For example, cDNA sequences encoding heavy chains and light chains can be cloned and recombined into GS expression vectors. The recombinant immunoglobulin expression vector can stably transfect CHO cells. As a more recommended prior art, mammalian expression systems will lead to glycosylation of antibodies, especially at the highly conserved N-terminal site of the Fc region. Stable pure strains are obtained by expressing antibodies that specifically bind to human LAG-3. Positive pure strains are expanded and cultured in serum-free medium in a bioreactor to produce antibodies. The culture fluid that secretes the antibodies can be purified by conventional techniques. For example, use A or G Sepharose FF column containing adjusted buffer for purification. Wash away non-specifically bound components. Then use pH gradient method to elute bound antibodies, detect antibody fragments with SDS-PAGE, and collect. Antibodies can be filtered and concentrated by conventional methods. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular screening and ion exchange. The obtained product needs to be immediately frozen, such as -70℃, or freeze-dried.

"Administering" and "treating" when applied to an animal, a human, an experimental subject, a cell, a tissue, an organ, or a biological fluid, refers to the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with an animal, a human, a subject, a cell, a tissue, an organ, or a biological fluid. "Administering" and "treating" may refer to, for example, treatment, pharmacokinetics, diagnostics, research, and experimental procedures. Treatment of cells includes contact of an agent with a cell, and contact of an agent with a fluid, wherein the fluid is in contact with a cell. "Administering" and "treating" also mean the in vitro and ex vivo treatment of, for example, a cell by an agent, a diagnostic, a binding composition, or by another cell. "Treatment" when applied to human, veterinary or research subjects refers to therapeutic treatment, prophylactic or preventive measures, research and diagnostic applications.

"Treatment" means administering an internal or external therapeutic agent, such as a composition comprising any of the binding compounds of the invention, to a patient who has one or more disease symptoms for which the therapeutic agent is known to have a therapeutic effect. Typically, the therapeutic agent is administered in an amount effective to alleviate one or more disease symptoms in the patient or population being treated, to induce regression of such symptoms or to inhibit the development of such symptoms to any clinically measurable degree. The amount of therapeutic agent effective to alleviate any specific disease symptom (also referred to as a "therapeutically effective amount") may vary according to a variety of factors, such as the patient's disease state, age and weight, the patient's health, the patient's behavior, the patient's diet, the time of administration, the route of administration, the rate of excretion, the combination of drugs, etc., and the ability of the drug to produce the desired therapeutic effect in the patient. Whether a disease symptom has been alleviated can be assessed by any clinical test method commonly used by physicians or other professional health care professionals to assess the severity or progression of the symptom. Although the embodiments of the present invention (e.g., treatment methods or products) may not be effective in alleviating every target disease symptom, they should reduce the target disease symptoms in a statistically significant number of patients as determined by any statistical test method known in the art, such as Student's t test, chi-square test, U test according to Mann and Whitney, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test.

An "effective amount" includes an amount sufficient to ameliorate or prevent a symptom or condition of a medical disease. An effective amount also means an amount sufficient to allow or facilitate diagnosis. The effective amount for a particular patient or veterinary subject may vary depending on factors such as the condition to be treated, the patient's general health, the route of administration and dosage, and the severity of side effects. An effective amount may be the maximum dose or dosing regimen that avoids significant side effects or toxic effects.

"Tm value" refers to the thermal denaturation temperature of the protein, that is, the temperature when half of the protein is unfolded. At this time, the spatial structure of the protein is destroyed, so the higher the Tm value, the higher the thermal stability of the protein.

Invention details:

The present invention obtains a stable pharmaceutical composition (preparation) comprising a LAG3 antibody or an antigen-binding fragment thereof, an acetate buffer or histidine salt, sucrose and polysorbate 80, and the pharmaceutical composition (preparation) is more suitable for administration.

Figure 1 shows the effect of humanized anti-LAG-3 antibody samples on enhancing the secretion of IL-2 cytokine by SEB-stimulated activated T lymphocytes. The data results show that LAG-3 humanized antibody candidates Hu229-013 and Hu303-005 can enhance the secretion of IL-2 cytokine by activated T lymphocytes to varying degrees, and there is a drug concentration dose effect.

Figure 2 shows the effect of humanized anti-LAG-3 antibodies on tumor volume in U-87MG tumor-bearing mice. The results showed that after 14 days of administration, LAG-3 antibodies Hu229-0136mpk and Hu303-0056mpk had a certain anti-tumor effect, with tumor inhibition rates of 27.25% (p<0.05) and 34.94% (p<0.01), respectively; there was a significant difference from the control group (p<0.001vshIGg)

Figure 3 is the CE purity trend chart of Hu229-013 antibody at 40℃.

Figure 4 is the IEC neutral peak trend diagram of Hu229-013 antibody at 40℃.

Figure 5 is the non-reduced CE trend graph of Hu303-005 antibody at 40℃.

Figure 6 is the main peak trend diagram of Hu303-005 antibody at 40℃ iCE.

Figure 7 is the trend chart of Hu303-005 vibration SEC results.

Figure 8 shows the fitting result of the difference between the IEC data of Hu303-005 antibody at 0℃ and 40℃.

Figure 9 is a fitting diagram of the difference between the CE purity data of Hu303-005 antibody at 0℃ and 40℃.

Figure 10 shows the iCE/CE/DLS fitting results of Hu303-005 antibody formulation at 25℃ and 40℃.

Implementation example

The present invention is further described in detail by the following examples. These examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

The experimental methods in the embodiments of the present invention that do not specify specific conditions are usually carried out under conventional conditions or under conditions recommended by raw material or product manufacturers. Reagents that do not specify specific sources are conventional reagents purchased on the market.

Example 1. Preparation of LAG-3 antigen antibody 1. Protein design and expression

SEQ ID NO：1 UniProt Lymphocyte activation gene 3 protein (human LAG-3, Uniprot No.: P18627) is used as the template of LAG-3 of the present invention to design the amino acid sequence of the antigen and detection protein involved in the present invention. Different tags can be optionally fused to the LAG-3 protein, and then cloned into pHr vector (self-produced) or pTT5 vector (Biovector, Cat#: 102762) or pTargeT vector (promega, A1410), and transiently expressed in 293 cells or stably expressed and purified in CHO-S to obtain the encoding antigen and detection protein of the present invention. The following LAG-3 antigens are all human LAG-3 unless otherwise specified. LAG-3 extracellular region with Flag tag: LAG-3-Flag, used to immunize mice

SEQ ID NO: 1

Note: The underlined part is the signal peptide, and the italic part is the Flag-tag.

SEQ ID NO：2 Full-length LAG-3: used for constructing LAG-3 overexpressing cell lines, immunizing mice and detecting

SEQ ID NO: 2

+胞內區 Annotation: Signal peptide + extracellular domain +

+ Intracellular region

SEQ ID NO：3 LAG-3-Fc is a fusion protein of the extracellular domain of LAG-3 and hIgG1 Fc, used for detection

SEQ ID NO: 3

Note: The underlined part is the signal peptide, the double underlined part is the linker, and the italic part is Fc.

SEQ ID NO：4 Fusion protein of LAG-3 extracellular domain and mIgG2a Fc: LAG-3-mFc, used for detection

SEQ ID NO: 4

Note: The underlined part is the signal peptide, the double underlined part is the linker, and the italic part is mFc.

2. Purification of LAG-3 related recombinant proteins, as well as purification of fusion tumor antibodies and recombinant antibodies 1) Purification steps of Flag-tagged LAG-3-Flag recombinant protein:

Centrifuge the sample at high speed to remove impurities and concentrate it to an appropriate volume. Use 0.5×PBS to equilibrate the flag affinity column and wash 2-5 times the volume of the column. Load the cell expression supernatant sample after impurities removal onto the column. Wash the column with 0.5×PBS until the A280 reading drops to the baseline. Wash the column with PBS to wash the impurities and collect them. Use 100mM glycine, pH3.0. to extract the target protein and collect it for subsequent in vitro activation and further purification.

2) Purification of fusion tumors, recombinant antibodies, and Fc fusion proteins

The cell expression supernatant samples were centrifuged at high speed to remove impurities, the fusion tumor expression supernatant was purified using a Protein G column, and the recombinant antibody and Fc fusion protein expression supernatant was purified using a Protein A column. The column was rinsed with PBS until the A280 reading dropped to the baseline. The target protein was extracted with 100mM acetic acid, pH 3.0, and neutralized with 1M Tris-HCl, pH 8.0. After the extracted sample was appropriately concentrated, it was further purified using a gel chromatography Superdex200 (GE) equilibrated with PBS, and the deaggregated peak was collected and packaged for later use.

Example 2. Preparation of anti-human LAG-3 fusion tumor monoclonal antibody 1. Immunity

Anti-human LAG-3 monoclonal antibodies were produced by immunizing mice. The experiments used SJL white mice, female, 6 weeks old (Beijing Weitonglihua Experimental Animal Technology Co., Ltd., Animal Production License No.: SCXK (Beijing) 2012-0001). Housing environment: SPF grade. After the mice were purchased, they were raised in the laboratory environment for 1 week, with a 12/12 hour light/dark cycle, a temperature of 20-25℃, and a humidity of 40-60%. The mice that have adapted to the environment were immunized according to the following scheme. The immunization antigen is the extracellular region of human LAG-3 with a Flag tag (SEQ ID NO: 1).

Immunization scheme A: Cross-immunization with TiterMax® Gold Adjuvant (Sigma Cat No.T2684) and Thermo Imject® Alum (Thermo Cat No.77161). The ratio of antigen to adjuvant (TiterMax® Gold Adjuvant) was 1:1, and the ratio of antigen to adjuvant (Thermo Imject® Alum) was 3:1, 50μg/head/time (first immunization), 25μg/head/time (boost immunization). The antigen was emulsified and vaccinated on days 0, 7, 14, 21, 28, 35, and 42. On day 0, 50μg/head of emulsified antigen was injected subcutaneously (SC) at multiple points. On day 7, 25μg/head was injected intraperitoneally (IP). On days 14, 28, 35, and 42, the antigen was injected dorsally or intraperitoneally according to the presence of dorsal masses and abdominal swelling. Blood was collected on days 21, 35, and 49, and the antibody titer in the mouse serum was determined by ELISA. After 7 immunizations, mice with high antibody titers in serum and titers close to the platform were selected for spleen cell fusion. Immunization was boosted 3 days before spleen cell fusion, and 50 μg/mouse of antigen solution prepared with physiological saline was injected intraperitoneally (IP).

Immunization scheme B: Immunize mice with QuickAntibody-Mouse5W (KX0210041). The ratio of antigen to adjuvant is 1:1, 25μg/mouse/time (first immunization/boost immunization). Antigen and adjuvant are quickly and thoroughly mixed and then inoculated on days 0, 21, and 35. On day 0, 25μg/mouse of antigen is injected into the calf muscle (IM) of the mouse. On days 21 and 35, 25μg/mouse is injected in the same way (determine whether the third immunization is performed based on the titer). Blood is collected on days 28 and 42, and the antibody titer in the mouse serum is determined by ELISA. Mice with high antibody titers in serum and titers close to the platform are selected for spleen cell fusion. Immunization was boosted 3 days before spleen cell fusion by intraperitoneal (IP) injection of 50 μg/head of antigen solution prepared with physiological saline.

2. Spleen cell fusion

The optimized PEG-mediated fusion step was used to fuse spleen lymphocytes with myeloma cells Sp2/0 cells (ATCC® CRL-8287 ^TM ) to obtain fused tumor cells. The fused tumor cells were resuspended in complete culture medium (DMEM culture medium containing 20% FBS, 1×HAT, and 1×OPI) at a density of 0.5-1×10 ⁶ /ml, and seeded in 96-well plates at 100μl/well. After incubation at 37°C and 5% CO ₂ for 3-4 days, 100μl/well of HAT complete culture medium was added and cultured for 3-4 days until a needle-like pure strain was formed. The supernatant was removed, and 200 μl/well of HT complete medium (RPMI-1640 medium containing 20% FBS, 1×HT and 1×OPI) was added. The cells were cultured at 37°C, 5% CO ₂ for 3 days before ELISA detection.

3. Screening of fusion tumor cells

According to the growth density of fusion tumor cells, the fusion tumor culture supernatant is tested by binding ELISA method. The cell supernatant of the positive wells tested by binding ELISA is subjected to cell blocking experiment. The wells with positive binding and blocking are promptly expanded and frozen for seed preservation and sub-selected two to three times until a single cell pure strain is obtained.

Each sub-selected cell needs to be tested by LAG-3 binding ELISA and cell blocking experiments. Through the above experimental screening, the fusion tumor pure strain is obtained, and the antibody is further prepared by serum-free cell culture method. The antibody is purified according to the purification example for use in the detection example.

4. Sequencing of pure strains of fusion tumor-positive cells

SEQ ID NO：5 The process of selecting sequences from positive fusion tumors is as follows. Collect fusion tumor cells in logarithmic growth phase, extract RNA using Trizol (Invitrogen, Cat No.15596-018) according to the kit instructions, and reverse transcribe using PrimeScript ^TM Reverse Transcriptase kit (Takara, Cat No.2680A). The cDNA obtained by reverse transcription is amplified by PCR using mouse Ig-Primer Set (Novagen, TB326 Rev.B 0503) and then sent to a sequencing company for sequencing. The amino acid sequences corresponding to the heavy chain and light chain DNA sequences of the obtained fusion tumor pure strain mAb229 are shown in SEQ ID NO: 5, 6 and SEQ ID NO: 7, 8: mAb229-VH

SEQ ID NO: 5

SEQ ID NO：6 mAb229-VL

SEQ ID NO: 6

SEQ ID NO：7 mAb303-VH

SEQ ID NO: 7

SEQ ID NO：8 mAb303-VL

SEQ ID NO: 8

The obtained positive pure strains were subjected to ELISA experiments for binding to human LAG-3 (results are shown in Table 2 for EC50 values of protein-level binding activity), ELISA experiments for binding to human LAG-3 overexpressing CHO-s cells (results are shown in Table 2 for EC50 values of cell-level binding activity), and experiments for blocking the binding of LAG-3 antigen to Daudi cells (results are shown in Table 2 for EC50 values of blocking activity), and their affinity with human LAG-3 protein was detected (results are shown in Table 3).

The data in Table 2 show that LAG-3 antibodies mAb229 and mAb303 have good binding activity with human LAG-3 protein. LAG-3 antibodies mAb229 and mAb303 have good binding activity with CHO-S cells overexpressing human LAG-3 full-length protein. LAG-3 antibodies mAb229 and mAb303 can significantly block the binding of human LAG-3 antigen to Daudi cells.

The data in Table 3 show that the LAG-3 antibodies mAb229 and mAb303 of the present invention have strong binding activity and affinity to human LAG-3 protein.

Example 3. Humanization of anti-human LAG-3 mouse fusion tumor monoclonal antibody mAb229

By comparing the IMGT human antibody heavy and light chain variable region germline gene database and MOE software, the heavy chain and light chain variable region germline genes with high homology to mAb229 were selected as templates, and the CDRs of the mouse antibody were transplanted into the corresponding human templates to form a variable region sequence of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The amino acid residues were determined and annotated by the Kabat numbering system.

1. Selection of humanized framework of fusion tumor pure strain mAb229

SEQ ID NO：21 The humanized light chain template of the mouse antibody mAb229 is IGKV1-39*01 and hjk4.1, the humanized heavy chain template is IGHV7-4-1*02 and hjh6.1, and the humanized variable region sequence is as follows: Hu229VH-CDR graft

SEQ ID NO: 21

SEQ ID NO：22 Hu229VL-CDR graft

SEQ ID NO: 22

Note: The sequence is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The italics in the sequence are FR sequences, and the underline is CDR sequences.

2. Template selection and reversion mutation design of fusion tumor pure strain mAb229, see Table 4 below:

Note: For example, I48V means that according to the Kabat numbering system, the I at position 48 is mutated back to V. Grafted means that the mouse antibody CDR is implanted into the human germline FR region sequence.

Note: This table shows the sequences obtained by combining various mutations. For example, Hu229-005 shows that there are two mutations on the humanized mouse antibody Hu229-005: light chain HumAb229_VL.1A and heavy chain HumAb229_VH.1. The same applies to other mutations.

SEQ ID NO：21 The specific sequence of mAb229 humanization is as follows: Hu229VH.1 (same as Hu229VH-CDR graft)

SEQ ID NO: 21

SEQ ID NO：23 Hu229VH.1A

SEQ ID NO: 23

SEQ ID NO：24 Hu229VH.1B

SEQ ID NO: 24

SEQ ID NO：25 Hu229VH.1C

SEQ ID NO: 25

SEQ ID NO：22 Hu229VL.1 (same as Hu229VL-CDR graft)

SEQ ID NO: 22

SEQ ID NO：26 Hu229VL.1A

SEQ ID NO: 26

SEQ ID NO：27 Hu229VL.1B

SEQ ID NO: 27

SEQ ID NO：28 Hu229VL.1C

SEQ ID NO: 28

Example 4. Humanization of anti-human LAG-3 mouse fusion tumor monoclonal antibody mAb303

By comparing the IMGT human antibody heavy and light chain variable region germline gene database and MOE software, the heavy chain and light chain variable region germline genes with high homology to mAb303 were selected as templates, and the CDRs of the mouse antibody were transplanted into the corresponding human templates to form a variable region sequence of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The amino acid residues were determined and annotated by the Kabat numbering system.

1. Selection of humanized framework of fusion tumor pure strain mAb303

SEQ ID NO：29 The humanized light chain template of the mouse antibody mAb303 is IGKV1-39*01 and hjk4.1, and the humanized heavy chain template is IGHV1-3*01 and hjh6.1. The humanized variable region sequence is as follows: Hu303VH-CDR graft

SEQ ID NO: 29

SEQ ID NO：30 Hu303VL-CDR graft

SEQ ID NO: 30

Note: The sequence is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The italics in the sequence are FR sequences, and the underline is CDR sequences.

2. Template selection and reversion mutation design of fusion tumor pure strain mAb303, see Table 6 below:

Note: For example, L46R means that the 46th position L is mutated back to R according to the Kabat numbering system. Grafted means that the mouse antibody CDR is implanted into the human germline FR region sequence.

The mutant sequence combinations are as follows:

Note: This table shows the sequences obtained by combining various mutations. For example, Hu303-005 shows that there are two mutations on the humanized mouse antibody Hu303-005: light chain HumAb303_VL.1A and heavy chain HumAb303_VH.1. The same applies to other mutations.

SEQ ID NO：29 The specific sequence of mAb303 humanization is as follows: Hu303_VH.1 (same as Hu303VH-CDR graft)

SEQ ID NO: 29

SEQ ID NO：31 Hu303_VH.1A

SEQ ID NO: 31

SEQ ID NO：32 Hu303_VH.1B

SEQ ID NO: 32

SEQ ID NO：33 Hu303_VH.1C

SEQ ID NO: 33

SEQ ID NO：30 Hu303_VL.1 (same as Hu303VL-CDR graft)

SEQ ID NO: 30

SEQ ID NO：34 Hu303_VL.1A

SEQ ID NO: 34

SEQ ID NO：35 Hu303_VL.1B

SEQ ID NO: 35

SEQ ID NO：36 Hu303_VL.1C

SEQ ID NO: 36

SEQ ID NO：37 Hu303_VL.1D

SEQ ID NO: 37

Example 5. Preparation of recombinant and humanized antibodies

The antibody uses the constant region of human heavy chain IgG4/light chain kappa combined with various variable regions, and makes a S228P mutation in the Fc segment to increase the stability of the IgG4 antibody. Other known mutations in this field can also be used to increase its performance.

SEQ ID NO：38 Relink constant area:

SEQ ID NO: 38

SEQ ID NO：39 Light chain constant area:

SEQ ID NO: 39

SEQ ID NO：40 The heavy chain amino acid sequence of Hu229-013 is:

SEQ ID NO: 40

SEQ ID NO：41 The light chain amino acid sequence of Hu229-013 is:

SEQ ID NO: 41

SEQ ID NO：42 The heavy chain amino acid sequence of Hu303-005 is:

SEQ ID NO: 42

SEQ ID NO：43 The light chain amino acid sequence of Hu303-005 is:

SEQ ID NO: 43

1. Molecular cloning of recombinant antibodies

After sequencing, the variable region encoding gene sequences were obtained from the positive antibody molecules obtained from the fusion tumor screening. The first and last primers were designed based on the sequence obtained by sequencing, and the sequenced gene was used as a template to construct the VH/VK gene fragments of each antibody through PCR, and then homologously recombined with the expression vector pHr (with signal peptide and hIgG4/hkappa constant region gene (CH1-FC/CL) fragment) to construct the recombinant antibody full-length expression plasmid VH-CH1-FC-pHr/VL-CL-pHr.

2. Molecular cloning of humanized antibodies

After humanized design, the antibody sequence is codon-optimized to generate a coding gene sequence with human codon preference. Primers are designed for PCR to construct each antibody VH/VK gene fragment, and then homologously recombined with the expression vector pHr (with signal peptide and hIgG4/hkappa constant region gene (CH1-FC/CL) fragment) to construct the humanized antibody full-length expression plasmid VH-CH1-FC-pHr/VL-CL-pHr.

3. Expression and purification of recombinant and humanized antibodies

Plasmids expressing the light and heavy chains of the antibody were transfected into HEK293E cells at a ratio of 1:1.2. After 6 days, the expression supernatant was collected, impurities were removed by high-speed centrifugation, and purified using a Protein A column. The column was rinsed with PBS until the A280 reading dropped to the baseline. The target protein was extracted with an acidic extraction solution of pH 3.0-pH 3.5 and neutralized with 1M Tris-HCl, pH 8.0-9.0. After the extracted sample was appropriately concentrated, it was further purified using a gel chromatography Superdex200 (GE) balanced with PBS to remove aggregates, collect the monomer peak, and package it for later use.

The following biochemical test method is used to verify the performance and beneficial effects of the antibody of the present invention.

Example 6: ELISA experiment of LAG-3 antibody binding to human LAG-3 protein

The binding ability of anti-LAG-3 antibodies is detected by ELISA experiments between antibodies and human LAG-3 proteins. LAG-3 fusion proteins with Fc or mFc tags are fixed to 96-well ELISA plates by binding to anti-Fc or mFc antibodies coated in the ELISA plate. The strength of the signal after the addition of the antibody is used to determine the binding activity of the antibody and LAG-3. The specific experimental method is as follows.

Dilute goat anti-human Fc antibody (Jackson Immuno Research, Cat No. 109-005-008) or goat anti-mouse Fc antibody (Sigma, Cat No. M3534-1ML) to a concentration of 2 μg/ml with PBS (Sigma, Cat No. P4417-100TAB) buffer at pH 7.4, add 50 μl/well to a 96-well ELISA plate, and place in a 37°C incubator for 2 hours. After discarding the liquid, add 200 μl/well of 5% skim milk (Bright skim milk powder) blocking solution diluted with PBS, incubate in a 37°C incubator for 2.5 hours or place at 4°C overnight (16-18 hours) for blocking. After the blocking, discard the blocking solution, wash the plate 5 times with PBST buffer (pH7.4 PBS containing 0.05% tweeen-20), add 50μl/well of LAG-3-Fc fusion protein (produced in-house, SEQ ID NO: 3) or LAG-3-mFc fusion protein (produced in-house, SEQ ID NO: 4) diluted to 1μg/ml with sample diluent (pH7.4 PBS containing 1% BSA), and incubate in a 37℃ incubator for 1 hour or at 4℃ overnight. After the incubation, discard the reaction solution in the ELISA plate, wash the plate 6 times with PBST, add 50μl/well of different concentrations of the test antibody (fusion tumor purified antibody or humanized antibody) diluted with sample diluent, and incubate in a 37℃ incubator for 1 hour. After incubation, wash the plate 5 times with PBST, add 100 μl/well of HRP-labeled goat anti-mouse secondary antibody (Jackson Immuno Research, Cat No.115-035-003) or goat anti-human secondary antibody (Jackson Immuno Research, Cat No.109-035-003) diluted with sample diluent, and incubate at 37°C for 1 hour. After washing the plate 6 times with PBST, add 50 μl/well of TMB colorimetric substrate (KPL, Cat No.52-00-03), incubate at room temperature for 5-15 minutes, add 50 μl/well of 1M H ₂ SO ₄ to terminate the reaction, read the absorbance at a wavelength of 450 nm using a NOVOStar enzyme labeler, and calculate the EC50 value of LAG-3 antibody binding to human LAG-3. The results are shown in Table 8. The data indicate that the humanized antibodies screened in the present invention have high binding activity to human LAG-3 protein.

Example 7: Binding experiment of LAG-3 antibody and humanLAG-3 overexpressing CHO-S cells

The binding ability of anti-LAG-3 antibodies was tested by binding experiments between antibodies and CHO-S cells overexpressing LAG-3 protein. The full-length LAG-3 plasmid (produced in-house, SEQ ID NO: 2) was transfected into CHO-S cells by electrofection and then pressure-screened for two weeks before the expression of LAG-3 was detected. After the overexpressed cells were fixed to the bottom of a 96-well plate, the strength of the signal after the addition of the antibody was used to determine the binding activity of the antibody and LAG-3 overexpressing CHO-S cells. The specific experimental method is as follows.

The cells were inoculated into a 96-well plate at a density of 4×10 ⁵ /ml and 100 μl/well for overnight culture. The supernatant was discarded, and the plates were washed three times with PBS. Then 100 μl/well of 4% PFA was added for fixation at room temperature for half an hour, and then washed three times with PBS. After the liquid was discarded, 200 μl/well of 5% skim milk (Bright skim milk powder) blocking solution diluted with PBS was added, and the plates were incubated at 37°C for 2.5 hours for blocking. After the blocking, discard the blocking solution, wash the plate 5 times with PBST buffer (pH7.4 PBS containing 0.05% tween-20), add 50μl/well of different concentrations of the test antibody (fusion tumor purified antibody or humanized antibody) diluted with sample diluent, and incubate in a 37℃ incubator for 1 hour. After the incubation, wash the plate 5 times with PBST, add 100μl/well of HRP-labeled goat anti-mouse secondary antibody (Jackson Immuno Research, Cat No.115-035-003) or goat anti-human secondary antibody (Jackson Immuno Research, Cat No.109-035-003) diluted with sample diluent, and incubate at 37℃ for 1 hour. After washing the plate six times with PBST, 50 μl/well TMB colorimetric substrate (KPL, Cat No. 52-00-03) was added and incubated at room temperature for 5-15 min. 50 μl/well 1M H ₂ SO ₄ was added to terminate the reaction. The absorbance was read at a wavelength of 450 nm using a NOVOStar enzyme labeler to calculate the EC50 value of the LAG-3 antibody binding to LAG-3 overexpressing CHO-S cells.

Example 8: Anti-LAG-3 antibody blocking the binding experiment of LAG-3 antigen and Daudi cells

Daudi cells (human leukemia cells, purchased from the Chinese Academy of Sciences Cell Bank) were inoculated at 3×10 ⁵ /well in a 96-well culture plate, centrifuged at 1000 rpm, the supernatant was discarded, and 4% PFA was added for fixation at room temperature for 30 minutes. After discarding the fixative, the plates were washed 4 times with PBS buffer, and 200 μl/well of 5% skim milk (Bright skim milk powder) diluted with PBS was added for blocking, and the plates were incubated at 37°C for 2.5 hours. After blocking, the blocking solution was discarded, and the plate was washed five times with PBST buffer (pH7.4 PBS containing 0.05% tweeen-20), and then 50 μl/well of a mixture of LAG-3-Fc fusion protein (produced in-house, SEQ ID NO: 3) labeled with biotin (biotin labeling kit, Dongren Chemical, Cat No. LK03) with a final concentration of 0.4 μg/ml and gradient concentrations of the antibody to be tested diluted with sample diluent (pH7.4 PBS containing 1% BSA) and pre-mixed and incubated for 1 hour was added, and the mixture was placed in a 37°C incubator for 1 hour. After incubation, discard the reaction solution in the ELISA plate, wash the plate with PBST for 5 times, add 50μl/well of HRP-labeled streptavidin (Sigma, Cat No. S2438) diluted with sample diluent, and incubate at 37°C for 1 hour. After washing the plate with PBST for 5 times, add 50μl/well of TMB colorimetric substrate (KPL, Cat No. 52-00-03), incubate at room temperature for 5-15min, add 50μl/well of 1M H ₂ SO ₄ to terminate the reaction, read the absorbance at a wavelength of 450nm using a NOVOStar ELISA reader, and calculate the blocking effect of LAG-3 antibody on the binding of antigen to daudi cells. The results are shown in Table 9. The data indicate that the humanized antibodies screened in the present invention can significantly block the binding of human LAG-3 antigen to Daudi cells.

Example 9: BIAcore detection of LAG-3 antibody affinity experiment

1. According to the method described in the instructions of the mouse anti-capture reagent kit (Cat.# BR-1008-38, GE), the mouse anti-capture antibody was covalently coupled to the CM5 biosensor chip (Cat.# BR-1000-12, GE) to capture the antibody to be tested by affinity, and then LAG-3-Flag (produced in-house, SEQ ID NO: 1) antigen was passed over the chip surface, and the reaction signal was detected in real time using the Biacore instrument to obtain the binding and dissociation curves, and the affinity values were obtained by fitting, see Table 2 above. After each cycle of dissociation in the experiment, the biochip was washed and regenerated with the regeneration solution configured in the mouse anti-capture reagent kit. The results showed that LAG-3 antibodies mAb229 and mAb303 had strong binding activity and affinity to human LAG-3 protein.

2. According to the method in the human antibody capture kit (Cat.# BR-1008-39, GE), the human anti-capture antibody was covalently coupled to the CM5 biosensor chip (Cat.# BR-1000-12, GE) to capture the antibody to be tested by affinity, and then LAG-3-Flag (produced in-house, SEQ ID NO: 1) antigen was passed through the chip surface. The reaction signal was detected in real time using the Biacore instrument to obtain the binding and dissociation curves, and the affinity values were obtained by fitting, as shown in Table 10 below. After each cycle of dissociation in the experiment, the biochip was washed and regenerated with the regeneration solution configured in the human anti-capture kit. The results show that the humanized antibodies screened by the present invention have strong binding activity and affinity to human LAG-3 protein.

Example 10: PBMC-T lymphocyte activation experiment

In order to study the activation of T lymphocytes by LAG-3 antibodies, human peripheral blood mononuclear cells (PBMCs) were collected and purified, and the superantigen Staphylococcus aureus enterotoxin B (SEB) was used to stimulate in vitro for 72 hours, and the secretion level of IL-2 cytokine was detected. The experimental process is briefly described as follows: Freshly separated and purified PBMCs were inoculated into 96-well cell culture plates with a cell density of about 1×10 ⁵ /well, and 100ng/mL SEB superantigen was added for stimulation. At the same time, gradient dilutions of antibody samples (diluted with culture medium) or culture medium were added as blank controls. After 72 hours of culture at 37℃, 5% CO ₂ incubator, the cell culture supernatant was collected. ELISA (BD, CAT# 550611) was used to detect the IL-2 secretion level in the cell culture supernatant. For specific operation, please refer to the reagent manual.

The results are shown in Figure 1. LAG-3 humanized antibody candidates Hu229-013 and Hu303-005 can enhance the secretion of cytokine IL-2 by activated T lymphocytes to varying degrees, and have a drug concentration dose effect.

Example 11: Effect of LAG-3 antibody on the inhibition of U-87MG subcutaneous transplanted tumors

This experiment was used to study the effect of humanized anti-LAG-3 antibodies on tumor volume in U-87 MG tumor-bearing mice.

Human brain glioma U-87 MG cells (3.5×10 ⁶ cells) were inoculated into the right rib of NOD-SCID mice (purchased from Changzhou Cavens Laboratory Animal Co., Ltd.) at 100μl. After the tumor grew to about 40mm ³ on day 10-14, the mice were randomly divided into three groups according to the tumor volume: control group, Isotype matched hIgG, LAG-3 humanized candidate antibody Hu229-013 group, and Hu303-005 group (grouping and dosage are shown in Table 11), with 8 mice in each group (D0). PBMC stimulated with CD3 antibody were injected into the tumor tissue at a volume of 5×10 ⁵ cells/60μl, and the antibody was injected intraperitoneally three times a week for a total of 6 times. Measure the tumor volume twice a week and record the data. The calculation formula for tumor volume (V) is: tumor volume (TV) = 1/2 × L _long × L _short ²

The tumor volume of each group of animals was expressed as mean ± standard deviation (Mean ± SEM) and plotted using Graphpad Prism 5 software. Two-way ANOVA was used for statistical analysis and the tumor inhibition rate was calculated using the formula: tumor proliferation rate (T/C %) = (TT ₀ /CC ₀ ) × 100%

Tumor inhibition rate %TGI=1-T/C %

The experimental results are shown in Table 11 and Figure 2. After 14 days of administration, LAG-3 antibodies Hu229-013 6mpk and Hu303-005 6mpk both had certain anti-tumor effects, with anti-tumor rates of 27.25% (p<0.05) and 34.94% (p<0.01), respectively; there was a significant difference from the control group (p<0.001 vs hIGg).

Note: D0: time of first drug administration; * p<0.05, ** p<0.01, *** p<0.001 vs hIGg, analyzed by two way ANOVA.

Example 12. Mouse PK assay of anti-LAG-3 humanized antibodies, Hu229-013 and Hu303-005

18 ICR mice, male, weighing 18-22 g, were purchased from Xipuer-Bikai Laboratory Animal Co., Ltd. During the feeding period, the mice were allowed free access to feed and water. They were acclimatized to the laboratory environment for no less than 3 days, with a 12/12 hour light/dark cycle, a temperature of 16-26°C, and a relative humidity of 40-70%. The day before the experiment, the ICR mice were numbered and randomly divided into groups, with 3 mice in each group. On the day of the experiment, the two groups of mice were intravenously injected with humanized candidate antibodies (Hu229-013) at a dosage of 3mg/kg and 10mg/kg; each mouse in the two groups was intravenously injected with humanized candidate antibodies (Hu303-005) at a dosage of 3mg/kg and 10mg/kg. The intravenous injection volume was 20ml/kg.

Blood was collected at 15 minutes, 8 hours, 1 day, 2 days, 4 days, 7 days, 10 days, 14 days, 21 days, 28 days, and 35 days after drug administration. About 0.1 ml of whole blood was collected each time without adding anticoagulant. After blood collection, it was placed at 4°C for 30 minutes, centrifuged at 1000g for 15 minutes, and the supernatant was placed in an EP tube and stored at -80°C.

The blood drug concentration in serum was detected by ELISA method, and Winnolin software was used to calculate the T1/2 and main parameters of the tested drug. The main pharmacokinetic parameters obtained are shown in Table 12:

The exposure levels of LAG-3 humanized antibodies Hu229-013 and Hu303-005 in mice are similar, and the exposure levels, peak concentrations and dose growth of the two antibodies at doses of 3 and 10 mg/kg are basically linearly related, showing linear kinetic characteristics.

Exemplary antibody pharmaceutical composition (preparation) preparation process

Step 1: The LAG-3 antibody stock solution is filtered through a 0.22μm PVDF filter. After filtering, the central control sample is tested for sterility and the filtrate is collected.

Step 2: Adjust the filling volume to 5.3ml, fill the filtered liquid into a 6ml vial, add a stopper, and take samples at the beginning, middle, and end of filling to detect the filling volume difference.

Step 3: Start the rolling and covering machine, add aluminum cover, and start rolling and covering.

Step 4: Visual inspection to confirm that the product has no defects such as inaccurate filling quantity. Print and paste the vial label; print the paper box label, fold the paper box, pack the box, and paste the paper box label.

Exemplary antibody pharmaceutical composition preparation process:

Step 1: Pass the Hu303-005 stock solution prepared in the prescription through a 0.22μm PVDF filter element. After filtration, take samples for sterility testing and collect the filtrate.

Step 2: Adjust the volume to 5.3ml, fill the filtered solution into a 20ml vial, half-fill with freeze-dried stopper, freeze-dry the original solution in the vial, and seal with rubber stopper.

Step 3: Start the rolling and covering machine, add aluminum cover, and start rolling and covering.

Step 4: Visual inspection to confirm that the product has no freeze-drying collapse or other defects. Print and paste the vial label; print the paper box label, fold the paper box, pack it, and paste the paper box label.

Example 13. Screening of LAG-3 Antibody Preparation Buffer System

In a series of buffers at 10mM pH5.0-7.5, LAG-3 antibody Hu229-013 or Hu303-005 preparations with a protein concentration of 50mg/mL were prepared. Each preparation was filtered, filled, stoppered, capped and sealed. The samples were subjected to forced degradation experiments such as high temperature and shaking at 40℃, and the appearance, molecular exclusion chromatography (SEC), non-reduced sodium dodecyl sulfate (CE-SDS)-capillary electrophoresis and ion exchange chromatography (IEC) or full-column imaging capillary isoelectric focusing (iCIEF) were used as evaluation indicators. The results are shown in Tables 13-1 and 13-2, and the statistical analysis results are shown in Figures 3 to 6.

Note: 0.01 mg/ml polysorbate 80 was added to the shaking D12 sample, and other samples did not contain polysorbate 80; D represents days.

Note: D means day; N/A means not tested.

The results show that:

(1) The Hu229-013 antibody has the best appearance in the acetic acid-sodium acetate (AA) system, followed by the succinic acid-sodium succinate (SA) and histidine-hydrochloric acid (His-HCl) systems. The acetic acid-sodium acetate (AA), pH 5.5, succinic acid-sodium succinate (SA), pH 6.0, citric acid-sodium citrate (CA), pH 6.0, and histidine-hydrochloric acid (His), pH 6.0 systems have higher CE and IEC purity at 40°C. Considering the appearance and CE and IEC results, the LAG-3 antibody Hu229-013 is more stable in the AA (pH 5.5), SA (pH 6.0), and His-HCl (pH 6.0) systems, as shown in Figures 3 and 4.

(2) The oscillation appearance data of Hu303-005 antibody showed that the appearance was better at lower pH, and the buffer systems were histidine-hydrochloric acid (His-HCl) and acetic acid-sodium acetate (AA). Under accelerated conditions of 40°C, CE-SDS (non-reducing) and iCIEF showed a significant decrease, among which CE-SDS data showed that pH 6.0 was better, and the buffer systems were acetic acid-sodium acetate, histidine-hydrochloric acid (His-HCl) and phosphate systems were better. iCE data showed that the neutral peak decreased less at higher pH, see Figures 5 and 6. Comprehensive consideration, the best buffer system is 10mM His-HCl pH6.0.

Example 14. Screening of excipients in LAG-3 antibody preparations

(1) In the buffer containing different concentrations of surfactant and sugar, LAG-3 Hu229-013 preparation with a protein concentration of 50 mg/mL, 10 mM succinate-sodium succinate, pH 6.0 was prepared. The results are shown in Table 14-1. The details are as follows:

1) 0.1mg/mL Polysorbate 20 (PS20)

2) 0.1mg/mL polysorbate 80 (PS80)

3) 70mg/mL sucrose

4) 70mg/mL trehalose

5) 50mg/mL mannitol

6) 50mg/mL sorbitol

(2) In buffer solutions containing different concentrations of surfactant and sugar, Hu303-005 antibody concentration was 50 mg/mL, containing 10 mM sodium acetate, pH 5.5. The results are shown in Figure 7 and Table 14-2.

1) 75mg/mL sucrose + 0.2mg/mL PS80

2) 75mg/mL trehalose + 0.2mg/mL PS80

3) 0.05mg/mL Polysorbate 20 (PS20)

4) 0.05mg/mL Polysorbate 80 (PS80)

5) 0.2mg/mL PS20

6) 0.2mg/mL PS80

7) 0.4mg/mL PS20

8) 0.4mg/mL PS80

Each preparation was filtered, filled, stoppered, capped and sealed. The samples were subjected to forced degradation experiments such as high temperature at 40°C, repeated freezing and thawing, and shaking. The results are shown in Table 14 and Figure 7.

Note: D stands for day.

Note: D stands for days.

The results show that:

(1) After shaking, the appearance of the PS80 group of Hu229-013 antibody preparation was better than that of the PS20 group. After one freeze-thaw, a small amount of particles appeared in the sucrose group, and a large amount of particles appeared in the trehalose group. There was no difference between the other data groups. Therefore, polysorbate 80 and sucrose are the preferred excipients.

(2) The appearance results of Hu303-005 antibody preparation showed that trehalose was slightly better, but the SEC results showed that sucrose was slightly better. The overall difference between sucrose and trehalose was not much. The appearance and SEC data showed that PS80 was better than PS20. Increasing the content of polysorbate can significantly improve the appearance and SEC stability, see Figure 7. Therefore, PS80 is better, and the PS concentration should be greater than 0.2mg/ml.

Example 15. Evaluation of LAG-3 antibody stability

(1) Prepare LAG-3 antibody Hu229-013 preparations with a protein concentration of 50 mg/mL, 60 mg/ml sucrose, and 0.4 mg/mL polysorbate 80 in the following different buffers: 1) 10 mM acetic acid-sodium acetate (AA) pH 5.5; 2) 10 mM histidine-acetic acid (His-AA) pH 6.0; 3) 10 mM histidine-hydrochloric acid (His-HCl) pH 6.0; 4) 10 mM succinic acid-sodium succinate (SA) pH 6.0.

(2). Prepare Hu303-005 preparations with a protein content of 50 mg/ml, 75 mg/ml sucrose, and 0.4 mg/ml PS80 in the following different buffers:

1) 10mM acetic acid-sodium acetate (AA) pH 5.5

2) 10mM histidine-hydrochloric acid (His-HCl) pH 6.0

3) 10mM histidine-hydrochloric acid (His-HCl) pH 6.5

Each preparation is filtered, filled, stoppered, capped and sealed. After the sample is prepared, it is placed at 25℃ or 4℃ to test its stability. The test items are appearance, SEC, IEC or iCE, CE-SDS (non-reduced).

Note: M3.5 means 3.5 months

Note: M means month, N/A means not tested.

The results show:

(1) LAG-3 antibody Hu229-013 is relatively stable in both 10mM AA pH5.5 and 10mM His-AA pH6.0 systems.

(2) After Hu303-005 antibody was placed at 4°C for 3 months, the CE of the His-HCl (pH 6.5) group decreased slightly, and the iCE of the AA pH 5.5 and His-HCl pH 6.5 groups changed slightly. The most stable condition was His-HCl (pH 6.0).

Example 16. Optimization of LAG-3 antibody formulation

In order to further optimize the type, pH, and ionic strength of the buffer, after fixing the Hu229-013 antibody concentration to 50 mg/ml, the DOE experimental design was performed using JMP software, and a series of prescriptions were obtained using the RSM model. By using the forced degradation method, IEC, CE (non-reduced), and microfluidic imaging (MFI) were used as evaluation indicators, and the least squares method was used to perform statistical analysis on the results. The DOE parameters are shown in Table 16, and the experimental prescriptions and results are shown in Tables 17 and 18.

Note: M1 represents a month, and D represents a day.

The forced degradation data were fitted, and the results showed that the stability of LAG-3 antibody Hu229-013 was better in 10-30mM acetic acid-sodium acetate (AA) buffer or histidine-acetic acid buffer (His-AA), pH5.2-5.8 system, and the best buffer system was 10-30mM acetic acid-sodium acetate (AA), pH5.5.

Example 17. Stability test of LAG-3 antibody preparation

In 10mM acetic acid-sodium acetate pH5.5 buffer, prepare LAG-3 Hu229-013 preparation with a protein concentration of 60mg/mL, 60mg/ml sucrose, and 0.4mg/mL polysorbate 80: filter the preparation, fill it, add a stopper, roll the cap, and seal it. After the sample preparation is completed, place it at 4℃ to investigate its stability. The test items are appearance, SEC, IEC, and CE-SDS (non-reducing).

The results showed that the Hu229-013 preparation could be kept stable for 9 months at 4°C.

Example 18. Optimization of LAG-3 antibody formulation

(1) Optimization of Hu229-013 formulation components

In order to further optimize the concentrations of protein, sucrose and polysorbate 80, the buffer solution was set to 10mM acetic acid-sodium acetate, pH 5.5, and the DOE experimental design was performed using JMP software. A series of prescriptions were obtained using the RSM model. The results were statistically analyzed using the least squares method using the forced degradation method and IEC and CE (non-reduced) as evaluation indicators. The DOE parameters are shown in Table 20, the test results are shown in Table 21, and the statistical analysis results are shown in Figures 8, 9 and Table 21.

The forced degradation data were fitted, and the results were as follows: The difference between the IEC data at 0 and 40°C was used for fitting, R ² >0.98, P<0.06, and the model was valid. The results are shown in Figure 8. The difference between the CE purity data at 0 and 40°C was used for fitting, R ² >0.99, P<0.05, and the model was valid. The results are shown in Figure 9. The 40℃ IEC fitting results show that the better prescription is: protein concentration 40-60mg/ml, sugar concentration 30-90mg/ml, PS80 concentration 0.4-0.5mg/ml; the 40℃ CE fitting results show that the better prescription is: protein concentration 50-80mg/ml, sugar concentration 30-90mg/ml, PS80 concentration 0.1-0.5mg/ml. Therefore, the optimal range is: protein concentration 50-60mg/ml, sucrose concentration 30-90mg/ml, PS80 concentration 0.4-0.5mg/ml.

(2). Optimization of Hu303-005 antibody preparation components

After fixing the sucrose concentration at 75 mg/ml, DOE experimental design was performed with 10 mM His buffer pH, protein concentration and polysorbate concentration as variables. A series of prescriptions were obtained by applying the RSM model. The prescriptions are shown in Table 22. By using the forced degradation method, iCIEF, CE (non-reduced) and DLS were used as evaluation indicators. The results were statistically analyzed using the least squares method. The results are shown in Table 23 and Figure 10.

The forced degradation data were fitted, and the iCIEF/CE/DLS at 25 and 40 degrees fit better, and the model was effective. The results are shown in Figure 10.

The results showed that under high temperature conditions, the particle size increased with increasing pH, the neutral peak also increased, and the iCIEF change rate slowed down with decreasing temperature. The CE data was optimal at pH 6.0. Combined with the previous experimental results (10mM His pH 6.0 is more stable), and the optimal pH is determined to be 6.0; 40-degree CE data show that the protein concentration is better at 45-60 mg/ml, so the optimal concentration is set at 50 mg/ml; combined with the results of the effect of polysorbate concentration on protein stability under various conditions and the polysorbate concentration screening results in other test examples (better when the concentration is greater than 0.2 mg/ml), the PS80 concentration is set at 0.3 mg/ml; for the Hu303-005 antibody liquid preparation, in this embodiment and other embodiments, iCIEF showed a significant decrease under high conditions, and it is considered to develop a freeze-dried preparation. In order to ensure better formability and appropriate osmotic pressure of the freeze-dried preparation, the sucrose concentration is set at 75mg/ml.

Example 19. Lyophilization of LAG-3 Antibody Preparation

Prepare a lyophilized preparation of Hu303-005 with a protein content of 50 mg/ml, 10 mM histidine-HCl, pH 5.5 or 6.0, 75 mg/ml sucrose, and 0.4 mg/ml PS80. The lyophilization procedure is as follows:

The samples were placed at 4℃ and 25℃ to examine their stability. Samples were taken at different time points and reconstituted with appropriate amount of injection water for testing. The results are shown in Table 25 and Table 26. The results show that the Hu303-005 preparation has no significant changes in various M3 indicators at 25℃ accelerated and 4℃ long-term, and has good stability.

At the same time, the stability of the Hu229-013 antibody preparation after freeze-drying was determined. A buffer containing 10mM acetic acid-sodium acetate at pH 5.5 was used to prepare an anti-LAG-3 antibody preparation with a LAG-3 antibody protein concentration of 50mg/ml, 75mg/ml sucrose, and 0.4mg/ml polysorbate 80. The antibody was filled into a 2mL vial at 1.1mL/bottle, placed in a freezer, and freeze-dried. The freeze-dried samples were compared before and after freeze-drying and the stability was investigated. The results showed that the quality of Hu229-013 antibody did not change before and after freeze-drying, and the freeze-dried preparation had good stability during storage.

Example 20. Optimization of freeze-drying process parameters

A preparation of Hu303-005 antibody with a content of 50 mg/ml, 10 mM histidine-hydrochloric acid, pH 6.0, 75 mg/ml sucrose, and 0.4 mg/ml PS80 was prepared for freeze drying. The collapse temperature of Hu303-005 was measured by freeze drying microscope to be about -19°C. The primary drying temperature is an important parameter of the freeze drying process, so the partition temperature of the primary drying process was carefully optimized. The freeze drying parameters are shown in Table 27, and the results are shown in Table 28. The appearance of the freeze dried powder cakes at each temperature met the requirements, but a small amount of particles appeared in the appearance after reconstitution at -5°C, so the primary drying partition temperature was set to -10°C.

*一次乾燥及二次乾燥時間根據具體批量及壓力升測試而定。 The final freeze-drying process is as follows:

*The time for primary drying and secondary drying is determined by the specific batch size and pressure rise test.

Example 21. Other optional formulations

The stable drug formulation provided by the present invention can also be any of the following stable combinations: (1) LAG-3 antibody Hu229-013 90 mg/ml, 80 mg/ml sucrose, 0.4 mg/ml polysorbate 80, 15 mM acetic acid-sodium acetate buffer, pH 5.5; (2) LAG-3 antibody Hu229-013 90 mg/ml, 80 mg/ml sucrose, 0.4 mg/ml polysorbate 80, 15 mM acetic acid-sodium acetate buffer, pH 6.5; (3) LAG-3 antibody Hu229-013 90 mg/ml, 50 mg/ml sucrose, 0.4 mg/ml polysorbate 80, 25 mM acetic acid-sodium acetate buffer, pH 5.5; (4) LAG-3 antibody Hu229-013 70 mg/ml, 50 mg/ml sucrose, 0.3 mg/ml polysorbate 80, 10 mM acetic acid-sodium acetate buffer, pH 5.5; (5) LAG-3 antibody Hu229-013 70 mg/ml, 80 mg/ml sucrose, 0.3 mg/ml polysorbate 80, 15 mM acetic acid-sodium acetate buffer, pH 5.2; (6) LAG-3 antibody Hu229-013 40 mg/ml, 75 mg/ml sucrose, 0.4 mg/ml polysorbate 80, 10 mM acetic acid-sodium acetate buffer, pH 6.0; (7) LAG-3 antibody Hu229-013 55mg/ml, 75mg/ml sucrose, 0.4mg/ml polysorbate 80, 10mM acetic acid-sodium acetate buffer, pH 5.7; (8) LAG-3 antibody Hu229-013 30mg/ml, 70mg/ml sucrose, 0.5mg/ml polysorbate 80, 10mM acetic acid-sodium acetate buffer, pH 5.5; (9) LAG-3 antibody Hu229-013 20mg/ml, 70mg/ml sucrose, 0.2mg/ml polysorbate 80, 10mM acetic acid-sodium acetate buffer, pH 5.4; (5) LAG-3 antibody Hu229-013 15 mg/ml, 85 mg/ml sucrose, 0.1 mg/ml polysorbate 80, 25 mM acetic acid-sodium acetate buffer, pH 5.6; (11) LAG-3 antibody Hu229-013 50 mg/ml, 85 mg/ml sucrose, 0.3 mg/ml polysorbate 80, 25 mM acetic acid-sodium acetate buffer, pH 5.8; (12) LAG-3 antibody Hu229-013 50 mg/ml, 75 mg/ml sucrose, 0.4 mg/ml polysorbate 80, 25 mM acetic acid-sodium acetate buffer, pH 6.0; (13) LAG-3 antibody Hu229-013 55 mg/ml, 90 mg/ml sucrose, 0.4 mg/ml polysorbate 80, 10 mM acetic acid-sodium acetate buffer, pH 5.3; (14) LAG-3 antibody Hu229-013 50 mg/ml, 90 mg/ml sucrose, 0.4 mg/ml polysorbate 80, 10 mM acetic acid-sodium acetate buffer, pH 5.0; (15) LAG-3 antibody Hu303-005 90 mg/ml, 75 mg/ml sucrose, 0.3 mg/ml polysorbate 80, 30 mM histidine-HCl buffer, pH 6.0; (16) LAG-3 antibody Hu303-005 90mg/ml, 75mg/ml sucrose, 0.3mg/ml polysorbate 80, 30mM histidine-HCl buffer, pH5.5; (17) LAG-3 antibody Hu303-005 75mg/ml, 75mg/ml sucrose, 0.3mg/ml polysorbate 80, 30mM histidine-HCl buffer, pH5.0; (18) LAG-3 antibody Hu303-005 1mg/ml, 75mg/ml sucrose, 0.3mg/ml polysorbate 80, 5mM histidine-HCl buffer, pH6.0; (19) LAG-3 antibody Hu303-005 70mg/ml, 30mg/ml sucrose, 0.3mg/ml polysorbate 80, 5mM histidine-HCl buffer, pH6.0; (20) LAG-3 antibody Hu303-005 50mg/ml, 60mg/ml trehalose, 0.3mg/ml polysorbate 80, 10mM histidine-HCl buffer, pH6.0; (21) LAG-3 antibody Hu303-005 50mg/ml, 90mg/ml trehalose, 0.3mg/ml polysorbate 80, 10mM histidine-HCl buffer, pH6.0.

<210> 2

<211> 525

<212> PRT

<213> Homo sapiens

<220>

<221> Peptide

<223> Full length LAG3

<400> 2

<210> 3

<211> 675

<212> PRT

<213> Artificial sequence

<220>

<221> Peptide

<223> Fusion protein of LAG-3 extracellular region and hIgG1 Fc

<400> 3

<210> 4

<211> 677

<212> PRT

<213> Artificial sequence

<220>

<221> Peptide

<223> Fusion protein of LAG-3 extracellular region and mIgG2a Fc

<400> 4

<210> 5

<211> 124

<212> PRT

<213> Rat

<220>

<221> Structural domain

<223> mAb229-VH

<400> 5

<210> 6

<211> 107

<212> PRT

<213> Rat

<220>

<221> Structural domain

<223> mAb229-VL

<400> 6

<210> 7

<211> 120

<212> PRT

<213> Rat

<220>

<221> Structural domain

<223> mAb303-VH

<400> 7

<210> 8

<211> 107

<212> PRT

<213> Rat

<220>

<221> Structural domain

<223> mAb303-VL

<400> 8

<210> 9

<211> 5

<212> PRT

<213> Rat

<220>

<221> Structural domain

<223> mAb229 HCDR1

<400> 9

<210> 10

<211> 17

<212> PRT

<213> Rat

<220>

<221> Structural domain

<223> mAb229 HCDR2

<400> 10

<210> 11

<211> 15

<212> PRT

<213> Rat

<220>

<221> Structural domain

<223> mAb229 HCDR3

<400> 11

<210> 12

<211> 5

<212> PRT

<213> Rat

<220>

<221> Structural domain

<223> mAb303 HCDR1

<400> 12

<210> 13

<211> 17

<212> PRT

<213> Rat

<220>

<221> Structural domain

<223> mAb303 HCDR2

<400> 13

<210> 14

<211> 11

<212> PRT

<213> Rat

<220>

<221> Structural domain

<223> mAb303 HCD3

<400> 14

<210> 15

<211> 11

<212> PRT

<213> Rat

<220>

<221> Structural domain

<223> mAb229 LCDR1

<400> 15

<210> 16

<211> 7

<212> PRT

<213> Rat

<220>

<221> Structural domain

<223> mAb229 LCDR2

<400> 16

<210> 17

<211> 9

<212> PRT

<213> Rat

<220>

<221> Structural domain

<223> mAb229 LCDR3

<400> 17

<210> 18

<211> 11

<212> PRT

<213> Rat

<220>

<221> Structural domain

<223> mAb303 LCDR1

<400> 18

<210> 19

<211> 7

<212> PRT

<213> Rat

<220>

<221> Structural domain

<223> mAb303 LCDR2

<400> 19

<210> 20

<211> 9

<212> PRT

<213> Rat

<220>

<221> Structural domain

<223> mAb303 LCDR3

<400> 20

<210> 21

<211> 124

<212> PRT

<213> Artificial sequence

<220>

<221> Structural domain

<223> Hu229VH.1

<400> 21

<210> 22

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<221> Structural domain

<223> Hu229VL.1

<400> 22

<210> 23

<211> 124

<212> PRT

<213> Artificial sequence

<220>

<221> Structural domain

<223> Hu229VH.1A

<400> 23

<210> 24

<211> 124

<212> PRT

<213> Artificial sequence

<220>

<221> Structural domain

<223> Hu229VH.1B

<400> 24

<210> 25

<211> 124

<212> PRT

<213> Artificial sequence

<220>

<221> Structural domain

<223> Hu229VH.1C

<400> 25

<210> 26

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<221> Structural domain

<223> Hu229VL.1A

<400> 26

<210> 27

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<221> Structural domain

<223> Hu229VL.1B

<400> 27

<210> 28

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<221> Structural domain

<223> Hu229VL.1C

<400> 28

<210> 29

<211> 120

<212> PRT

<213> Artificial sequence

<220>

<221> Structural domain

<223> Hu303_VH.1

<400> 29

<210> 30

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<221> Structural domain

<223> Hu303_VL.1

<400> 30

<210> 31

<211> 120

<212> PRT

<213> Artificial sequence

<220>

<221> Structural domain

<223> Hu303_VH.1A

<400> 31

<210> 32

<211> 120

<212> PRT

<213> Artificial sequence

<220>

<221> Structural domain

<223> Hu303_VH.1B

<400> 32

<210> 33

<211> 120

<212> PRT

<213> Artificial sequence

<220>

<221> Structural domain

<223> Hu303_VH.1C

<400> 33

<210> 34

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<221> Structural domain

<223> Hu303_VL.1A

<400> 34

<210> 35

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<221> Structural domain

<223> Hu303_VL.1B

<400> 35

<210> 36

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<221> Structural domain

<223> Hu303_VL.1C

<400> 36

<210> 37

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<221> Structural domain

<223> Hu303_VL.1D

<400> 37

<210> 38

<211> 327

<212> PRT

<213> Artificial sequence

<220>

<221> Structural domain

<223> Recombinant protein constant region, S228P mutation.

<400> 38

<210> 39

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<221> Structural domain

<223> kappa light chain constant area

<400> 39

<210> 40

<211> 451

<212> PRT

<213> Artificial sequence

<220>

<221> CHAIN

<223> Heavy chain amino acid sequence of Hu229-013

<400> 40

<210> 41

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<221> CHAIN

<223> Light chain amino acid sequence of Hu229-013

<400> 41

<210> 42

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<221> CHAIN

<223> Heavy chain amino acid sequence of Hu303-005

<400> 42

<210> 43

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<221> CHAIN

<223> Light chain amino acid sequence of Hu303-005

<400> 43

This representative diagram does not include component symbols and their meanings.

Claims (26)

A pharmaceutical composition comprising (a) 1 mg/ml to 90 mg/ml of an anti-LAG-3 antibody or an antigen-binding fragment thereof, (b) 5 mM to 30 mM of acetic acid-sodium acetate buffer or histidine-hydrochloric acid buffer, pH 5.0-6.5, (c) 60 mg/ml to 90 mg/ml of sucrose or trehalose; (d) 0.02 mg/ml to 0.8 mg/ml of polysorbate 80; wherein the heavy chain amino acid sequence of the anti-LAG-3 antibody is the sequence shown in SEQ ID NO: 42, and the light chain amino acid sequence is the sequence shown in SEQ ID NO: 43. The pharmaceutical composition as described in item 1 of the patent application, wherein the concentration of the anti-LAG-3 antibody or its antigen-binding fragment is 40 mg/ml to 60 mg/ml. The pharmaceutical composition as described in item 2 of the patent application, wherein the concentration of the anti-LAG-3 antibody or its antigen-binding fragment is 50 mg/ml. The pharmaceutical composition as described in item 1 of the patent application, wherein the pH of the acetic acid-sodium acetate buffer or the histidine-hydrochloric acid buffer is 5.0 to 6.0. The pharmaceutical composition as described in item 1 of the patent application scope, wherein the concentration of the acetic acid-sodium acetate buffer or the histidine-hydrochloric acid buffer is 10mM to 30mM. The pharmaceutical composition as described in item 1 of the patent application, wherein the sugar is sucrose. The pharmaceutical composition as described in Item 1 of the patent application, wherein the concentration of sucrose or trehalose is 75 mg/ml. The pharmaceutical composition as described in item 1 of the patent application, wherein the concentration of polysorbate 80 is 0.2 mg/ml to 0.6 mg/ml. The pharmaceutical composition as described in Item 8 of the patent application, wherein the concentration of polysorbate 80 is 0.3 mg/ml to 0.5 mg/ml. The pharmaceutical composition as described in claim 1, comprising the components as shown in i) or ii) below: i) (a) 1 mg/ml to 90 mg/ml of an anti-LAG-3 antibody or an antigen-binding fragment thereof, (b) 5 mM to 30 mM acetic acid-sodium acetate buffer, pH 5.0-6.5, (c) 60 mg/ml to 90 mg/ml of sucrose and (d) 0.02 mg/ml to 0.8 mg/ml of polysorbate 20. or ii) (a) 1 mg/ml to 90 mg/ml of an anti-LAG-3 antibody or an antigen-binding fragment thereof, (b) 5 mM to 30 mM of histidine-hydrochloric acid buffer, pH 5.0-6.5, (c) 60 mg/ml to 90 mg/ml of sucrose or trehalose and (d) 0.05 mg/ml to 0.6 mg/ml of polysorbate 80; wherein the heavy chain amino acid sequence of the anti-LAG-3 antibody is the sequence shown in SEQ ID NO: 42, and the light chain amino acid sequence is the sequence shown in SEQ ID NO: 43. The pharmaceutical composition as described in item 1 of the patent application, comprising: (e) 40 mg/ml to 80 mg/ml of anti-LAG-3 antibody or antigen-binding fragment thereof, (f) 10 mM to 30 mM acetic acid-sodium acetate buffer, pH 5.2-5.8, (g) 70 mg/ml to 80 mg/ml of sucrose and (h) 0.4 mg/ml to 0.5 mg/ml of polysorbate 80; or the pharmaceutical composition Comprising: (e) 45 mg/ml to 60 mg/ml of an anti-LAG-3 antibody or an antigen-binding fragment thereof, (f) 10 mM to 30 mM of histidine-hydrochloric acid buffer, pH 5.5-6.0, (g) 60 mg/ml to 90 mg/ml of sucrose or trehalose and (h) 0.2 mg/ml to 0.6 mg/ml of polysorbate 80; wherein the heavy chain amino acid sequence of the anti-LAG-3 antibody is the sequence shown in SEQ ID NO: 42, and the light chain amino acid sequence is the sequence shown in SEQ ID NO: 43. The pharmaceutical composition as described in item 1 of the patent application, wherein the pharmaceutical composition comprises: (i) 50 mg/ml of anti-LAG-3 antibody or antigen-binding fragment thereof, (j) 10 mM histidine-hydrochloric acid buffer, pH 6.0, (k) 75 mg/ml sucrose, (l) 0.3 mg/ml polysorbate 80; wherein the heavy chain amino acid sequence of the anti-LAG-3 antibody is the sequence shown in SEQ ID NO: 42, and the light chain amino acid sequence is the sequence shown in SEQ ID NO: 43. A method for preparing a lyophilized preparation containing an anti-LAG-3 antibody or an antigen-binding fragment thereof, comprising the step of freeze-drying the pharmaceutical composition described in any one of items 1 to 12 of the patent application scope. A method for preparing a freeze-dried preparation containing an anti-LAG-3 antibody or an antigen-binding fragment thereof as described in Item 13 of the patent application, wherein the freeze-drying comprises the steps of pre-freezing, primary drying and secondary drying in sequence. A method for preparing a lyophilized preparation containing an anti-LAG-3 antibody or an antigen-binding fragment thereof as described in Item 14 of the patent application, wherein the primary drying temperature is -5°C to -20°C. A method for preparing a lyophilized preparation containing an anti-LAG-3 antibody or an antigen-binding fragment thereof as described in item 14 of the patent application, wherein the primary drying temperature is -10°C. A lyophilized preparation containing an anti-LAG3 antibody or an antigen-binding fragment thereof, wherein the lyophilized preparation can form a pharmaceutical composition as described in any one of items 1 to 12 of the patent application scope after reconstitution. A method for preparing a reconstituted solution containing an anti-LAG-3 antibody or an antigen-binding fragment thereof, comprising the step of reconstituted the lyophilized preparation described in claim 17. As described in item 18 of the patent application, the solvent used for reconstitution is water for injection. A reconstituted solution containing an anti-LAG-3 antibody or an antigen-binding fragment thereof prepared by the method described in claim 18. The reconstituted solution of the anti-LAG3 antibody or antigen-binding fragment thereof as described in claim 20, comprising the following components: i) (a) 40 mg/ml to 80 mg/ml of the anti-LAG-3 antibody or antigen-binding fragment thereof, (b) 10 mM to 30 mM acetic acid-sodium acetate buffer, pH 5.2-5.8, (c) 70 mg/ml to 80 mg/ml of sucrose, and (d) 0.4 mg/ml to 0.5 mg/ml of poly(acrylonitrile). Sorbitol 80; or ii) (a) 45 mg/ml to 60 mg/ml of an anti-LAG-3 antibody or an antigen-binding fragment thereof, (b) 10 mM to 30 mM of histidine-hydrochloric acid buffer, pH 5.5-6.0, (c) 60 mg/ml to 90 mg/ml of sucrose or trehalose, and (d) 0.2 mg/ml to 0.6 mg/ml of polysorbate 80; wherein the heavy chain amino acid sequence of the anti-LAG-3 antibody is the sequence shown in SEQ ID NO: 42, and the light chain amino acid sequence is the sequence shown in SEQ ID NO: 43. A use of a pharmaceutical composition as described in any one of items 1 to 12 of the patent application, or a lyophilized preparation as described in item 17 of the patent application, or a reconstituted solution as described in item 20 or 21 of the patent application, which is used as a medicine. A use of a pharmaceutical composition as described in any one of items 1 to 12 of the patent application, or a lyophilized preparation as described in item 17 of the patent application, or a reconstituted solution as described in item 20 or 21 of the patent application, for preparing a drug for treating a disease or condition related to LAG-3, wherein the disease or condition is cancer. The use as described in claim 23, wherein the cancer is selected from ovarian cancer, melanoma, prostate cancer, intestinal cancer, gastric cancer, esophageal cancer, breast cancer, lung cancer, kidney cancer, pancreatic cancer, uterine cancer, liver cancer, bladder cancer, cervical cancer, oral cancer, brain cancer, testicular cancer, skin cancer, thyroid cancer and hematological malignancies. The use as described in item 24 of the patent application, wherein the hematological malignant tumor includes myeloma, chronic leukemia and acute leukemia. A product comprising a container containing the pharmaceutical composition described in any one of items 1 to 12 of the patent application, the lyophilized preparation described in item 17 of the patent application, or the reconstituted solution described in item 20 or 21 of the patent application.

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